Abstract: The by-products of mandarin fish were hydrolyzed with alcalase, while its optimized preparing process was performed with the single factor and response surface experiments, which were based on the degree of hydrolysis. Additionally, the in vitro digestion of prepared hydrolysates was further analyzed. The results showed that: The optimum condition of the hydrolysis processing was that the ratio of solid-liquid was 1:4 g/mL, the pH was 9.1, the temperature was 57.0 ℃, the enzyme dosage was 2.02%, and the hydrolysis time was 4 h, with the degree of hydrolysis of which was 18.38%. During the simulated gastric digestion and the simulated intestinal digestion, the content of soluble peptides respectively decreased by 17.76% and 50.97%. Furthermore, after the whole gastrointestinal digestion, DPPH and ABTS radical scavenging rate, respectively, survived with values of 51.36% and 48.67%, which indicated that the prepared hydrolysates exerted relatively higher bioaccessibility. The results suggest that the by-products of mandarin fish might be utilized as a candidate for developing functional foods.