We explored the improved method to prepare decellularized kidney scaffold and provide experimental basis for kidney tissue engineering and renal pathology and toxicology in vitro research. We perfused rat kidneys with PBS (group control) and prepared the decellularized kidney scaffolds with sodium dodecyl sulfate (SDS) (group S), Triton X-100 combined with SDS (group TS), and Triton X-100 combined with SDS after repeated freezing and thawing (group FTS) in different flow velocity. Meanwhile we measured their fluid distributions and vascular resistance. We examined the degree of decellularization of acellular scaffolds by HE, DAPI staining and DNA quantification. We examined the retention of main composition and structural integrity of decellularized scaffolds by Masson, PAS and immunohistochemical staining. We also detected the ultrastructure, cytotoxicity and the level of growth factor of the scaffolds by scanning electron microscope, MTT and ELISA, respectively. The results showed that the time of decellularization in group FTS was less than that in group S and TS. The vascular resistance of scaffolds decellularized at 10 mL/min flow velocity was lower. The fluid distribution in groups S, TS and FTS was different from that in control group. No residual cell was detected by HE and DAPI staining. DNA content was less than 50 ng/mg. Masson, PAS and immunohistochemical staining results showed that there was extracellular collagen, polysaccharide, type I collagen, type IV collagen, fibronectin and laminin in the decellularized scaffolds, and the scanning electron microscope result showed the scaffolds had the honeycomb structure. The cytotoxicity level of decellularized scaffolds was between grade 0 to 1. The level of VEGF, EGF, IGF-1 and PDGF-BB in group FTS were significantly higher than those in group S and TS. In concluding, combining freeze-thawing with perfusion can produce more ideal and effective whole organ decellularized scaffold of rat kidney, and make a foundation for the study of kidney tissue engineering and in vitro pathology and toxicology of kidney.