A novel serine protease, ABUSV-SPase, was isolated to homogeneity for the first time from Chinese Agkistrodon blomhoffii ussurensis snake venom, and its enzymatic and structural properties were characterized by multiple techniques. ABUSV-SPase is a stable monomeric protein with a molecular mass of 26,752.6 a.m.u. It reacts optimally with its substrate Nα-tosyl-L-arginine methyl ester (TAME) at pH 7.0 and 41 °C. ESI-MS/MS analysis indicates that ABUSV-SPase is a new serine protease, sharing peptide homologies with various snake venom serine proteases, especially the snake venom thrombin-like enzymes of this group, and serine protease precursors. It is a zinc-containing protein, and although zinc is not essential for activity, its replacement by various divalent metal ions, including Mg2+, Mn2+, and Ca2+, increases the TAME hydrolysis activity of the enzyme. The intrinsic fluorescences of Tyr and Trp residues of ABUSV-SPase have emission wavelengths red-shifted by 12.8 nm and 3.6 nm from those of free Tyr and Trp, respectively. The zinc ion increases the hydrophobicity of the environment of the Trp residues, increases the thermostability of the protein, and affects the protein secondary structure to stabilize the enzyme, but appears to have no direct role in its esterase hydrolysis activity.