Protein preparation, which has active ingredients designated for the use of biomaterials and therapeutical protein, is obtained by genetic engineering, but products of genetic engineering are often contaminated by endotoxins. Because endotoxin is a ubiquitous and potent proinflammatory agent, endotoxin removal or depletion from protein is essential for researching any biomaterials. In this study, we have used Tris-acetate (TA) buffer of neutral pH value to evaluate endotoxins absorbed on the Pierce high-capacity endotoxin removal resin. The effects of TA buffer on pH, ionic strength, incubation time as well as human-like collagen (HLC) concentration on eliminating endotoxins are investigated. In the present experiments, we design an optimal method for TA buffer to remove endotoxin from recombinant collagen and use a chromogenic tachypleus amebocyte lysate (TAL) test kit to measure the endotoxin level of HLC. The present results show that, the endotoxins of HLC is dropped to 8.3 EU/ml at 25 mM TA buffer (pH 7.8) with 150 mM NaCl when setting incubation time at 6 h, and HLC recovery is about 96%. Under this experimental condition, it is proved to exhibit high efficiencies of both endotoxin removal and collagen recovery. The structure of treated HLC was explored by Transmission Electron Microscopy (TEM), demonstrating that the property and structure of HLC treated by TA buffer are maintained. Compared to the most widely used endotoxin removal method, Triton X-114 extraction, using TA buffer can obtain the non-toxic HLC without extra treatment for removing the toxic substances in Triton X-114. In addition, the present study aims at establishing a foundation for further work in laboratory animal science and providing a foundation for medical grade biomaterials.