In order to study secretory expression and properties of acid protease from Aspergillus japonicus and its application in pork tenderization, the acid protease gene (AjproA1) from Aspergillus japonicus was efficiently expressed in Pichia pastoris. After high cell-density fermentation in a 5L fermenter, the enzyme activity and protein concentration of the fermentation supernatant were 669.6U/mL and 6.64mg/mL, respectively. According to the results of amino acid multiple sequence alignment analysis, the acid protease belongs to the aspartic protease A1 family, which had the highest homology (73%) with acid protease from Aspergillus phoenicis, and was a novel acid protease. The acid protease was purified by strong anion exchange chromatography to obtain electrophoresis-grade pure enzyme. After purification, the optimal catalytic conditions for recombinant acid protease (AjproA1) was pH 3.0 and reaction temperature 45℃, and it had good stability in the pH range of 3.0-6.0 and temperature below 40℃. The results of the hydrolysis analysis of this acid protease for different proteins showed that the enzyme exhibited broad substrate hydrolysis specificity and the highest hydrolysis activity toward κ-casein for casein components. AjproA1 was used to tenderize pork and effectively reduced the shearing force of pork to achieve a good tenderization effect. The research results aimed to provide a theoretical reference for the development of novel acid proteases and their application in meat tenderization.