The availability of carbon (C), nitrogen (N), and other substrates in soil determines the growth and metabolism of microorganisms and affects the activity of extracellular enzymes. To study the activities of -1,4-glucosidase (BG) and -1,4--acetylglucosaminidase (NAG) in response to C and N availability, samples that underwent four treatments-non-fertilization (CK), chemical fertilizer (NPK), combination of organic manure and chemical fertilizer (OM), and mixture of straw and chemical fertilizer (ST)-were collected from long-term fertilization paddy soil and incubated for 0, 4, 8, and 12 months to obtain soil with different C and N availability gradients. The results showed that the dissolved organic carbon(DOC) content of OM and ST treatment samples was 2-3 times higher than that of CK and NPK treatment samples. With the increase of DOC and ammonium (NH-N) contents, the activities of BG and NAG and the contents of microbial biomass C (MBC) and N (MBN) showed no increase during incubation within each treatment. Fertilization treatments, incubation time, and their interaction are crucial factors varying the contents of DOC, NH-N, MBC, and MBN among different fertilization treatments (<0.01). There was a positive correlation between MBC/MBN and DOC/NH-N of OM treatment (<0.05) and a negative relationship between ln(BG)/ln(NAG) and DOC/NH-N of ST treatment (<0.01), indicating that the availability of substrates played a key role in the potential activity of extracellular enzymes in paddy soil, and the carbon-nitrogen ratio of microbial biomass was controlled by the C/N stoichiometry of substrates in soil. The results have a certain guiding significance for further study on the variation of extracellular enzyme activity in paddy soil, regulating the balance of carbon and nitrogen, and improving the fertility of paddy soil.