The objective of this study is to innovatively evaluate the biochemistry performance of α-chitosan from Portunus trituberculatus shell and β-chitosan from Illex argentinus squid gladius by using the weighted composite index method, and provide a theoretical basis for better development and utilization of chitosan biomedical materials. To build a composite evaluation system, seven key indicators, including molecular weight (Mw), deacetylation degree (DD), water binding capacity (WBC), fat binding capacity (FBC), thermal stability (TS), primary structure and secondary structure, which significantly affect chitosan biochemical characteristics, were determined and analyzed. The viscosity average Mw of chitosan was in the range of 22.5–377.1 kDa, and the DD was 83.4–97.8%. Thermogravimetric (TG) and differential scanning calorimetry (DSC) analyses of commercial chitosan (CS), crab chitosan (CSC) and squid chitosan (CSS) showed a downward trend in TS, while WBC and FBC showed an obvious upward trend. FT-IR had a similar profile in peak shape, but the peak position slightly shifted. CD indicated that chitosan maintained the double helix structure and multiple secondary structural elements. The composite weighted index values of CS, CSC and CSS were 0.85, 0.94 and 1.31 respectively, which indicated that the CSS biochemistry performance was significantly better than CSC, and β-chitosan has great potential in biomedical materials.