[Objective] The paper was to establish ultrafiltration membrane technology for separation of antitumor active plypeptides from Periplaneta Americana. [Method] Hollow fiber ultrafiltration membrane(3 kDa)was used to separate antitumor active plypeptides from P. Americana. Through single factor test, the impact of rotation speed, concentration ratio, pH and operation time on separation effort was studied, and the separation technology conditions were further optimized through orthogonal test. [Result] The best technology conditions for separation of antitumor active plypeptides from P. Americana were as follows: rotation speed 4 500 r/min, concentration ratio 30%, pH 11, and the rotation speed was the major influencing factor. [Conclusion] The study laid foundation for industrialization of ultrafiltration membrane separation of antitumor active plypeptides from P. Americana. [Objective] The paper was to establish ultrafiltration membrane technology for separation of antitumor active peptides from Periplaneta Americana. [Method] Hollow fiber ultrafiltration membrane (3 kDa) was used to separate antitumor active peptides from P. Americana. Through single factor test, the impact of rotation speed, concentration ratio, pH and operation time on separation effort was studied, and the separation technology conditions were further optimized through orthogonal test. [Result] The best technology conditions for separation of antitumor active plypeptides from P. Americana were as follows : rotation speed 4 500 r / min, concentration ratio 30%, pH 11, and the rotation speed was the major influencing factor. [Conclusion] The study laid foundation for industrialization of ultrafiltration membrane separation of antitumor active plypeptides from P. Americana.