The 17-year-old Kiwi???s round was the joint-lowest 18-hole score in the history of the Ladies European Tour and at one point she admitted to having designs on a 59.
Teeing off on the back nine, Ko opened with a quartet of birdies to reach the turn in just 30 strokes.
She then suffered her only bogey of the day at 10 before recovering immediately to make an eagle-three at the 424-yard 11th.
Ko ended her round with five birdies in the last six holes at Clearwater Golf Club and she said: “I didn’t know what I was shooting but when I went past the scoreboard on 13 I thought, oh, 59, that is the magical number. But I’m pretty happy with 61.”
Ko joins six other players who share the record for the all-time lowest score on Tour: Trish Johnson, Kirsty Taylor, Nina Reis, Lisa Holm Sorensen, Karrie Webb and So Yeon Ryu.
Her nearest challenger after 36 holes is another teenager, England???s Charley Hull, who posted a 64 to stand at 10-under-par and is alone in second spot.
Hull, who has made just a solitary bogey in two days, admitted that getting to grips with the greens has been the hardest part.
The 18-year-old said: “Coming from Royal Melbourne last week the greens were so fast and really tricky.
???With these ones you can be a bit firmer and not allow so much break. I think I had 26 putts. I hit some good putts out there so I’m pretty happy with the way I played.”
Scores after 36 Holes:
-13: Lydia Ko (Nzl) (61)
-10: Charley Hull (Eng) (64)
-9: Giulia Sergas (Ita) (67)
-9: Beth Allen (USA) (67)
-8: Marianne Skarpnord (Nor) (69),
-8: Pamela Pretswell (Sco) (67)
-8: Nanna Koerstz Madsen (Den) (69)
-8: Marina Alex (USA) (67)
-6: Amelia Lewis (USA) (65)
-6: Marta Sanz (Spa) (67)
-6: Laura Davies (Eng) (67)
-6: Hannah Green (Aus) (70)
-6: Felicity Johnson (Eng) (68)