To increase uniform deposition of nano-hydroxyapatite (n-HAP) in porous chitosan (CS) scaffolds, an alkaline phosphatase (ALP) enzyme-induced biomineralization method was developed. The results showed that beta-glycerol phosphate calcium (beta-GPC) could enter the scaffolds slowly through dialysis, be further decomposed to calcium and phosphate ions by preexisting ALP inside the scaffolds, and finally be transformed into n-HAP after aging for certain amount of time. The XRD and FT-IR results verified the formation of weakly crystalline HAP and showed that higher concentrations of ALP and more aging time were beneficial for HAP formation. These n-HAP minerals were observed to be densely and uniformly deposited on the CS by SEM. Thermogravimetric analysis revealed improved thermal stability of the composites with the increase of inorganic content. Notably, the scaffolds promoted osteogenic differentiation of pre-osteoblasts in vitro and demonstrated excellent tissue integration in vivo. The unique biomineralization method developed herein might provide a novel strategy for fabricating bioactive degradable composites as bone replacement materials.