This study provides an enzymatic kinetic resolution (EKR) method for the biocatalytic enantioselective synthesis of chiral β‐keto acetates. For the first time, the attitude of several lipase enzymes has been examined under different conditions for the EKR of the racemic 1,3‐keto alcohols. We found that the Amano lipase from Pseudomonas fluorescens (AL‐PF) lipase showed the best results for the resolution of the racemic 1,3‐keto alcohols. We developed a highly effective EKR method by changing the enzyme, temperature, solvent, and enzyme amount in order to synthesize the chiral 1,3‐keto esters, which are useful intermediates in organic synthesis. This study presents an efficient method for the synthesis of 1,3‐keto acetates with a good yield and high enantioselectivity with the R configuration.