In the extraction of collagens from mammalian tissues, the free pepsin used in the acid–pepsin extraction system is hard to recycle, and there is a risk of enzyme protein contamination in the extracted collagen products, which limits their applications. To solve this problem, an immobilized pepsin was successfully prepared via the covalent crosslinking of glutaraldehyde using a 3-aminopropyltriethoxysilane (APTES) surface modified silica clay as the support. The immobilized pepsin was applied for the extraction of collagen from bovine hide. The optimal immobilization process involves incubating pepsin with an initial concentration of 35 mg mL⁻¹ and glutaraldehyde with 5% activated APTES modified silica clay at 25 °C for 60 min, by which the loading amount of pepsin was 220 mg g⁻¹ and the activity of the immobilized pepsin was 4.2 U mg⁻¹. The collagen extracted using acetic acid and the immobilized pepsin method retained its complete triple helix structure. This research thus details an effective separation method using pepsin for extraction of collagen via an acetic acid–enzyme method, where the extracted collagen may be a candidate for use in biomaterial applications.