The commonly used methods to produce diosgenin by acid hydrolysis generate large amounts of wastewater and other pollution. Exploring a new environmentally friendly method is becoming urgent. Mild conditions and less pollution are the advantages of biotransformation. Therefore, aiming at exploring the effect of probiotic Lactobacillus on the production of diosgenin via biotransformation, batch studies were performed. Twenty-four strains maintained in laboratory were screened by conversion rate and pH. Based on screening, purification and 16 s rDNA sequences, four probiotic strains were identified and classified. The results indicated that all the strains had biotransformation potential. Among all the strains, four strains, including L20, L35, L83 and L33, performed better, with conversion rates reaching up to 15.12%, 15.83%, 17.07% and 17.47%, respectively. The highest conversion rate, 17.47%, was that of L33 and the lowest one, 7.07%, of L4. Nineteen of the 24 strains achieved more than 10% conversion rate. The pH decreased from 7 to 3-4 after fermentation, reflecting the progress of fermentation and indicating the end of the biotransformation process. Molecular characterization based on 16S rDNA homology of partial sequences confirmed the preliminary identification as L. rhamnosus (L20, L35), L. paracasei (L83) and L. salivarius (L33).