Abstract: Objective　To investigate the two-step enzymatic method for the detection of serum creatinine. Methods　　The two-step enzymatic method for the detection of serum creatinine was established using creatinine amidohydrolase as the reagent Ⅱ and N-(2-hydroxy-3-sulfopropyl)-3，5-dimethoxyaniline sodium salt（HDAOS）and 4-mino-antipyrine (4-AAP) as the reagent Ⅰ. The inner blank method was used to eliminate endogenous creatine, and altered spectral absorption method was used to eliminate the interference of lipemia, hemolysis and jaundice. The levels of serum creatinine in 36 patients were determined by the two-step enzymatic method. The results were compared with those of high performance liquid chromatography (HPLC) and single-reagent method. The serum creatinine levels of 200 healthy subjects were determined and performed as reference range.  Results　There was significant difference between two-step enzymatic method[creatinine：（84.2±26.6）μmol/L] and single-reagent method [creatinine：(116.6±29.6)μmol/L, t=32.12，P＜0.01]，whereas there was no statistical significance between two-step enzymatic method and HPLC [creatinine：（83.9±26.8）μmol/L, t=0.541 6，P＞0.05], and showed good correlation (Y_{two-step enzymatic method} =1.042X_HPLC－0.182 1，r²＝0.982 4). The liner range of the two-step enzymatic method for the detection of serum creatinine was up to 4 500 μmol/L,the average rate of recovery was 100.8％，and the within-run coefficient of variation (CV) and between-run CV were 2.91%-4.20% and 3.20%-4.60%. The sera [low level：78.0 μmol/L, middle level：206.0 μmol/L and high level：900.0 μmol/L] were determined by the two-step enzymatic method for 180 d. The interday CV were 4.46%, 5.27% and 7.24%. The reagents of the two-step enzymatic method could be stable at least for 180 d. The reference range of serum creatinine in healthy males was 56-132 μmol/L, and that in healthy females was 41-109 μmol/L.  Conclusions　The two-step enzymatic method based on creatinine amidohydrolase as reagent Ⅱ and based on HDAOS and 4-AAP as reagent Ⅰ could eliminate the interference of creatine，lipemia，hemolysis and jaundice, and it can be applied as routine test.

Key words: Creatinine, Two-step enzymatic method, Inner blank method, N-（2-hydrroxt-3-sulfopropyl）-3, 5-dimethoxyaniline sodium salt, Endogenous interference