Fulvic acid (FA) is an important component of soil humus. As an intermediate substance in soil humification, the structural characteristics of FA play an important role in indicating the improvement of soil organic matter. The combined organic and inorganic fertilizers are an effective measure for soil fertility improvement, straw resource utilization and inorganic fertilizer reduction. In order to explore the effect of straw organic fertilizer instead of inorganic fertilizer (nitrogenous fertilizer) on soil FA in the black soil region of Heilongjiang Province, six treatments were set up, including no fertilization (CK), a single application of inorganic fertilizer (NPK), 25% of organic nitrogen fertilizer (NPKM1), 50% (NPKM2), 75% (NPKM3), and 100% (NPKM4). The contents of soil organic carbon (SOC) and FA were determined. The source of soil FA was characterized by fluorescence index (FI) and biological index (BIX), and the degree of soil humification was analyzed by humification index (HIX). Three-dimensional fluorescence spectrum parallel factor analysis method was used to analyze the fluorescence components and maximum fluorescence intensity (F_max) of soil FA, and redundancy analysis (RDA) was used to explore the response relationships among fluorescence intensity, soil organic carbon and different treatments. The results showed that compared with NPK treatment, the contents of SOC and soil FA increased significantly in the treatments of combined application of organic and inorganic fertilizers, the greatest impact on NPKM2 treatment, SOC and soil FA content increased by 8.06% and 13.84%. Soil FA was affected by both autochthonous and terrestrial sources (FI>1.4, 0.8F_max values of fulvic-acid-like and humic-acid-like first increased and then decreased, and the F_max value of protein-like gradually decreased. The F_max values of fulvic-acid-like and humic-acid-like were the highest, and the relative percentage of humic-acid-like was the highest. The results of RDA showed that NPKM2 treatment had the greatest effect on the content of SOC and soil FA. Therefore, based on the analysis of soil FA fluorescence spectrum characteristics, in order to improve the content of soil organic matter, increase straw utilization rate and reduce the application of inorganic fertilizer, the treatment of straw organic fertilizer replacing inorganic nitrogen fertilizer by 50% was the best organic-inorganic fertilizer ratio.