Abstract: Biodegradability is a crucial indicator to evaluate the ecological properties of leather. Herein, a variety of tanned leathers were employed as the test objects, and their biodegradability in an aqueous medium was determined by the ISO 20136 standard by analysis of evolved CO₂. The degradation conditions were optimized. The results showed that the depickled hide could replace type I collagen as the reference material. When the sample-to-activated sludge mass ratio was 1∶3, the sample mass concentration was 0.2-0.6 g/L, and the degradation temperature was 30 ℃, the difference between the biodegradability of various types of leather could be observed after degradation for 20 days. The biodegradability of the chrome-free tanned leathers used in this study was better than that of chrome tanned leather. The widespread application of this biodegradability determination method is expected to provide support for the green and sustainable development of leather industry.