Straw and biochar, two commonly used soil amendments, have been shown to enhance soil fertility and the composition of microbial communities. To compare the effects of straw and biochar on soil fertility, particularly focusing on soil dissolved organic matter (DOM) components, and the physiochemical properties of soil and microbial communities, a combination of high-throughput sequencing and three-dimensional fluorescence mapping technology was employed. In our study, we set up four treatments, i.e., without biochar and straw (B0S0); biochar only (B1S0); straw returning only (B0S1); and biochar and straw (B1S1). Our results demonstrate that soil organic matter (SOM), available nitrogen (AN), and available potassium (AK) were increased by 34.71%, 22.96%, and 61.68%, respectively, under the B1S1 treatment compared to the B0S0 treatment. In addition, microbial carbon (MBC), dissolved organic carbon (DOC), and particulate organic carbon (POC) were significantly increased with the B1S1 treatment, by 55.13%, 15.59%, and 125.46%, respectively. The results also show an enhancement in microbial diversity, the composition of microbial communities, and the degree of soil humification with the application of biochar and straw. Moreover, by comparing the differences in soil fertility, DOM components, and other indicators under different treatments, the combined treatments of biochar and straw had a more significant positive impact on paddy soil fertility compared to biochar. In conclusion, our study revealed the combination of straw incorporation and biochar application has significant impacts and is considered an effective approach to improving soil fertility.