In order to screen myrosinase-producing lactic acid bacteria, palladium chloride assay was used to determine the myrosinase enzyme activity, and the myrosinase-producing strains were screened from 39 strains of plant-derived lactic acid bacteria. The screened strains were identified by morphological characteristics, physiological and biochemical experiments in combination with 16S rDNA sequence analysis, and the enzyme-producing conditions of the strains were optimized by using the single-factor experiment and response surface method. The results showed that three strains of lactic acid bacteria(named as B244, 161 and 602, respectively) could effectively degrade glucosinolates in the growth environment. Strain B244 was identified as Pediococcus pentosaceus, and 161 and 602 were both identified as Limosilactobacillus fermentum. Pediococcus pentosaceus had a strong ability to produce myrosinase, and the optimal culture conditions for the myrosinase production were as follows: 4.2 mmol/L glucosinolates was added to LB medium, the mass concentration of peptones was adjusted to 14.3 g/L with the initial pH of 6.9 and incubation time of 16 h at 40 ℃. The myrosinase activity of B244 under the optimized condition reached 117.2 U/L, which was 52.9% higher than that(76.65 U/L) before optimization.