Objective To discuss the value of laparoscopic biopsy of retroperitoneal lymph nodes in the diagnosis of diseases.Methods Six consecutive patients with enlargement of retroperitoneal lymph nodes underwent laparoscopic exploration and retroperitoneal lymph node biopsy between June 2000 and December 2002. One of the patients also underwent enterolysis. Results A total of 1～3 lymph nodes were removed respectively in the 6 patients, with a diameter of 0 5～1 2 cm. Pathological diagnosis included 1 case of primary peritonitis, 1 case of tuberculous peritonitis accompanying lymphoid tuberculosis, 3 cases of lymphoma, and 1 case of chronic lymphocytic leukemia. There were no postoperative complications, but 1 case died of MSOF originated from primary disease on the 13th postoperative day. Conclusions Laparoscopic biopsy of retroperitoneal lymph nodes offers a safe and effective alternative in the diagnosis of enlargement of retroperitoneal lymph nodes.