Ko, who hasn't won on the LPGA Tour since last April, sits just two shots off leaders Yu Liu of China and South Korean world No. 1 Sung-Hyun Park.
World No. 2 Ariya Jutanugarn of Thailand sits eight shots off the pace in a tie for 43rd, while Australian world No. 3 Minjee Lee narrowly made the cut but has plenty of ground to make up sitting at one-under in a tie for 70th place.
First-round leader Celine Boutier sat with Ko in a tie for third after a poor end to her second round. After scoring an eagle on the par-5 15th, holing out from a greenside bunker, the Frenchwoman added three shots to her card over the final three holes to lose her place at the top of the leaderboard.
Meanwhile, Danny Lee avoided the cut at the Valspar Championship in Florida.
A second round even-par 71 has seen Lee sit at one-over for the tournament, just one shot ahead of the cut.
Paul Casey and Austin Cook lead at six-under par.