Abstract: In this study, the mucosa of porcine small intestine was used as the raw material to optimize the optimal process for the extraction of heparin (Hep) by high hydrostatic pressure enzymatic hydrolysis by response surface test, to study the adsorption conditions of Hep purified by macroporous resin, and to explore the stability of Hep (pH, temperature, and light). The results showed that the optimal enzymatic process was alkaline protease, 3.1 h, 55 ℃, pH9.5, pressure 100 MPa, enzyme addition 9%, salt concentration 2.0%, the actual average concentration of Hep was measured to be 11.56±0.12 U/mL. The purification of Hep was carried out by using the D958 resin, with the up-sampling flow rate of 1.5 BV/h, the concentration of the eluent of 4 mol/L NaCl, and the elution flow rate of 1.5 BV/h, the purity of Hep could reach 68.75% after purification, and the electrophoretic purity achieved. In the stability study, Hep had the best stability at pH7.5, better stability at 40 ℃, and light had no effect on the stability of Hep. This study can greatly improve the extraction effect of Hep and provide theoretical and technical support for the high value processing and utilization of pig small intestine.