The invention relates to the technical field of free fatty acid determining, in particular to a free fatty acid determining kit. The kit comprises a reagent R1 and a reagent R2, wherein the R1 is prepared from coenzyme A, triphosadenine, acyl coenzyme A synthetase, MgCl2, a Trinder substrate and Tween-20, and has the pH of 7.2; the R2 is prepared from flavin adenine dinucleotide, 4 amino-antipyrine, peroxidase, acyl coenzyme A oxidase, Tween-20, glycerinum, 1 to 1.5g/L of brij35-1 and ethylene diamine tetraacetic acid disodium salt and has the pH of 7.2. By adding different combinations of protein protecting agents, the enzyme activity stability is prolonged; the 37-DEG C stability of the enzyme is prolonged from 7 days to 10 days; the enzyme denaturation is slowed down; the reagent R2 cannot easily generate precipitate.