Abstract: To address the low bioavailability of oyster peptide (OP) after gastrointestinal digestion, this study optimized and investigated the preparation process and colloidal properties of chitosan-oyster peptide nanoparticles (CONP). The CONP were characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction, with evaluations of their stability, simulated digestion characteristics, and release kinetics. Results indicated that the optimal preparation conditions were: Chitosan (CS) to sodium tripolyphosphate (TPP) mass ratio of 4.8:1, CS concentration of 2.4 mg/mL, CS to OP mass ratio of 1:2, and CS solution pH of 5.5. Under these conditions, the encapsulation efficiency reached 85.44%, with a particle size of 259.67 nm. The CONP formed amorphous nanoparticles primarily through electrostatic and hydrogen bonding interactions, exhibiting irregular elliptical shapes with geometric dimensions around 100 nm and uniform particle size distribution. The peak thermal decomposition temperature of encapsulated OP increased by 37 °C. After treatment with 200 mmol/L NaCl, a 90 °C water bath for 120 min, and pH 2 PBS, the particle sizes of CONP were 709, 338, and 431 nm, respectively. After 360 min of continuous simulated gastrointestinal digestion, the cumulative release rate reached 70.80%. After 24 h of separate simulated gastric and intestinal digestion, the release rate in gastric fluid was significantly lower than in intestinal fluid. The formation mechanism and structural characteristics of CONP significantly enhanced its ionic strength stability, thermal stability, and pH responsiveness. It exhibited high stability in simulated gastric fluid following Fickian diffusion, while showing swelling behavior and following Super Case II release in simulated intestinal fluid, with rapid release of OP. This study provides a theoretical foundation for the application of OP in functional food development.