Isopyrazam (IPZ) is a new chiral fungicide. For bioactivity, there was a 3.37-1578 times difference among the four stereoisomers. For Alternaria alternata and Phoma multirostrata, cis-(1S,4R,9S)-IPZ had the greatest activity. Using cis-IPZ might improve the efficacy and reduce the dosage of the racemate by 54.7-72.2% for A. alternata and P. multirostrata. To zebrafish, trans-IPZ showed the highest acute toxicity (LC50, 0.096 mg/L). The degradation half-lives of IPZ stereoisomers in the five crops ranged from 3.50 to 15.2 days. Cis-IPZ was preferentially degraded in grape, pear, and celery. The residual concentrations of IPZ in grape and celery were still higher than the maximum residue limit, and the acute and chronic dietary intake risks of IPZ in celery were unacceptable (RQa: 146-250%, HQ: 117-200%), which were worthy of further researching. Based on the research results, it is safer and more reasonable to use IPZ in the form of a racemate with a high ratio of cis-IPZ.