Compositions comprising thrombin and methods of preparing them are disclosed. The composition may be in the form of an aqueous solution at pH 5.7-7.4 consisting essentially of 0.1 mg/mL to 5.0 mg/mL thrombin, 2% to 4% (w/v) sucrose, 3.5% to 5% (w/v) mannitol, 50 mM to 300 mM NaCl, 0-5 mM CaCl.sub.2, 0.03% to 1% (w/v) of a surfactant or high-molecular-weight polyethylene glycol, and a physiologically acceptable buffer, or may be in lyophilized form.