Squid pen (SP) was found to contain 64.41% protein and 26.03% chitin. The amino acid composition revealed that Met was the most abundant amino acid in SP, with a concentration of 13.67 g/100 g. To enhance the stability and bioavailability of SP hydrolysates, microcapsules were developed using ultrasonic emulsification techniques with SP trypsin hydrolysates (SPTH) and SP β-chitosan (SPC). The optimal preparation conditions involved using a 2% concentration of SPC, a 4 mg/mL concentration of SPTH, a core-to-wall ratio (/) of 1:3 for SPTH/SPC, and subjecting them to ultrasonic treatment for 20 min. These microcapsules had a loading capacity of 58.95% for SPTH under these conditions. The successful encapsulation of SPTH in the SPC complex to form SPC-SPTH microcapsules was confirmed by FTIR, XRD, DSC, and SEM, exhibiting good thermal stability, small particle size, and high encapsulation efficiency. In vitro digestion studies demonstrated a release of 15.61% in simulated gastric fluid and 69.32% in intestinal fluid, achieving targeted release in the intestines. The digested products exhibited superior antioxidant activity compared to free SPTH digests, suggesting that microencapsulation effectively preserves SPTH bioactivity. This study enhances the bioavailability of SPTH and offers a promising delivery system for natural compounds with low bioavailability and stability.