The world number 44 became the first player to win consecutive fifth singles rubbers in the final after outclassing Lajovic, ranked down at 117 in the world, in what was undoubtedly the biggest match of his 23-year-old opponent's career.
Stepanek, who beat Nicolas Almagro in four sets in the fifth match of last year's final - in which Czech Republic defeated Spain 3-2 - comfortably outplayed the young Serb to recorded a 6-3 6-1 6-1 triumph.
Earlier on Sunday, Novak Djokovic made sure the final went to a fifth rubber against the defending champions as he saw off Tomas Berdych.
The world number two claimed a 6-4 7-6 (7-5) 6-2 victory over the 28-year-old in two hours and 42 minutes to level the contest at 2-2.
The Czech Republic took a 2-1 lead on Saturday when Stepanek and Berdych needed only three sets to beat Iliya Bozoljac and Nenad Zimonjic 6-2 6-4 7-6 (7/4).
But on Sunday and in front of his home crowd, the 2013 Australian Open winner ensured the match would go to a final rubber as Serbia attempt to wrestle back the title they won in 2010.
Lajovic, winner of the 2012 Samarkand Challenger, was a late replacement for the hosts after Janko Tipsarevic was ruled out due to injury on Thursday night.
Tipsarevic was forced to admit defeat in his battle to recover from a heel injury sustained in Valencia in October, denting Serbian hopes of winning a second title after their 2010 final triumph over France.
Stepanek took a 4-1 lead in the first set before the young Serb pulled it back to 4-2 and then 5-3, but the 34-year-old went on to claim a 6-3 win and leave Czech Republic two sets away from retaining the title.
Lajovic looked like he was struggling to read what Stepanek was going to do and fell 4-0 behind in the second set, working hard to return as his opponent had him running all over the court.
The Serbian won the following game but his errors were pilling up and it was going to require a mammoth effort to come back after the Czech took the next two games to put the visitors two sets up.
The 34-year-old Stepanek, who also broke his own record as the oldest player to win a live fifth rubber in Davis Cup history, took the first game before Lajovic held his serve to make it 1-1.
Djokovic and the Serbia team were willing their man on from the sidelines but with telling experience over his young opponent, Stepanek made it look almost easy as he won the next four games.
The crowd were still getting behind Lajovic as the match was drawing to a conclusion but his defensive lob went long.
Stepanek then put away a forehand and a big serve out wide before a clinical smash made it 6-1 and handed the Czechs their second consecutive title.
Stepanek told British Eurosport: "I don't think words can describe it.
"It's completely different than the first time and to defend this trophy means the world for us right now, for our country.
"We waited 32 years for our second title and now we are one of the five countries who've been able to defend it.
"And we did it playing in Serbia on their surface in front of their fantastic crowd, but thanks to our fans who've been with us no matter where we played we made history today."
In the open era, the only previous defending champions to retain their title were Spain in 2009, West Germany in 1989, Sweden in both 1985 and 1998 and the United States on several occasions.
On becoming the first man in Davis Cup final history to win the live fifth rubber two years running, Stepanek said: "That's a beautiful stat, what can I say?
"I came to this match confident because I have the experience, I felt great on the court here since Friday and I just proved it and I'm indescribably happy."
Asked where the victory stood among his career achievements, he added: "Definitely the highest.
"Both of the years. To win the Davis Cup is so far my biggest achievement.
"I got in the top 10 in singles, the top 10 in doubles, I won two grand slams (in doubles), and played the Masters in singles and doubles - but winning two Davis Cups tops them all."
He was fully appreciative of the travelling support for the Czech Republic team, and praised them after Sunday's match.
He said: "From the guys who are supporting us very loudly, I know every one personally and I will thank them for the great support they gave us and we are proud of them.
"I hope the country will be proud of us because we're a very small country and to make such an achievement is really very good."