[Objective] Cow dung is commonly used as organic manure in agricultural production. It comes up to be an important issue whether application of cow manure coupled with biochar or biochar-amended cow manure could increase net carbon transformation rate of the organic materials in soil as to high-efficient use of organic materials.[Method] In this study, a one-year field experiment was conducted to explore effect of application of biochar amended cow manure on net carbon transformation rate and structure of humic substances (HS) in soil by comparison. The experiment was designed to have four treatments, that is, M1 (cow manure at a normal rate), M2 (cow manure twice the normal rate), CBM1(M1 amended with biochar) and CBM2(M2 amended with biochar).[Results] Results show that compared with Treatment M1 and M2, Treatment CBM2 increased the content of humic acid (HA), fulvic acid(FA) and humin (Hu) up to 1.36 g·kg^-1, 2.50 g·kg^-1 and 5.03 g·kg^-1, respectively, and the net carbon transformation rate of HA and FA by 1.18% and 3.67%. Compared with the application of cow manure alone, the application of cow manure amended with biochar was much higher in relative content of aliphatic functional groups and polysaccharide in HA, FA and Hu. The amendment of biochar increased the relative content of aromatic functional groups of HA and Hu in Treatment CBM2, but decreased that of FA in Treatment CBM1.[Conclusion] All the findings show that CBM treatments are better than M treatments, forming more HS. Moreover, it also favors formation of aliphatic and polysaccharide functional groups. Biochar amendment increases the aromatic function groups of HA and Hu. In the treatments amended with biochar Hu and FA increases in condensation degree, while decreases in oxidation degree, but HA responds reversely.