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The extraction of enzyme preparations from bovine pancreas is a key step in the produc-tion of pancreatin used for pharmaceutical and food industry applications. However, conventional methods (СМ) often fail to preserve enzymatic activity during processing, particularly under variable temperature and pH conditions. This study investigates the potential of ultrasound-assisted extraction (UAM) as an alternative to CM for improving the recovery, stability, and performance of two essential pancreatic enzymes—α-amylase (AA) and protease (PA). Enzymatic activities were assessed over a broad temperature range (10–50 °C) and pH spectrum (5.5–8.0), with both methods evaluated under identical conditions. UAM consistently yielded higher enzymatic activity across all tested pa-rameters, with optimal AA and PA observed at pH 6.0 and 38 °C. Notably, UAM-extracted enzymes retained significant activity even at elevated temperatures (46–50 °C), whereas CM-derived samples showed marked loss of function. These findings demonstrate that UAM enhances enzyme release and thermal resilience by minimizing denaturation and structural degradation during extraction. The results support the use of UAM as a scalable, efficient strategy for producing stable pancreatin preparations suitable for industrial and therapeutic use.