The determination of organic nitrogen (N) mineralization is crucial for estimating N availability, quantifying exogenous inputs, and estimating associated environmental impacts. The objective of this study was to explore the effect of long-term various fertilization on soil organic N mineralization potential (NMP), which influences plant N accessibility. Treatments from a 26-year long-term field experiment with no fertilization (CK), chemical fertilizer N at 165 kg N ha−1 and P at 82.5 kg P2O5 ha−1 (NP), NP with K fertilizer at 165, 82.5, 82.5 kg ha−1 N, P2O5 and K2O (NPK), NPK at 165, 82.5, 82.5 kg ha−1 N, P2O5 and K2O with manure at 7857.14 kg ha−1 (NPKM), and NPKM at 165, 82.5, 82.5 kg ha−1 N, P2O5 and K2O with manure at 1.5× application rate (11,785.71 kg ha−1) (1.5NPKM) were examined for potentially mineralizable N by aerobic incubation at 35 °C for 30 weeks. Three pools (Pools I, II, and III) of mineralizable N were recognized. Pool I, the mineralization flush on rewetting in the first 2 weeks; Pool II, gross N mineralization between weeks 2 and 30; and Pool III, the potentially mineralizable N, predicted from the fitted curve, that did not mineralize during the incubation period. Soil microbial biomass carbon (SMBC) and N (SMBN) as well as fixed ammonium (NH4+) contents and relationship with N mineralization rate (k) were also studied. Long-term manure application yielded a significantly higher k (0.32 week−1) than other treatments (0.12–0.22 week−1) but not a significantly higher NMP. Nitrogen mineralization during the wheat and maize-growing seasons was predicted to be 8.7–26.3 (mg N kg−1 soil) and 25.9–42.1 (mg N kg−1 soil), respectively. Both labile mineralizable N pools (Pools I and II) followed the same patterns in the treatments: 1.5NPKM > NPKM > NPK > NP > CK, while the reverse was true for stable N (Pool III). The significant positive correlation between k with SMBC and SMBN (R2 = 0.93, p = 0.008 and R2 = 0.94, p = 0.006) suggested that the higher mineralization rate might be contributed by the higher soil microbial biomass in NPKM. The trends of fixed NH4+ and mineralized N were coupled. Long-term manure application significantly improved the N mineralization rate in soil. Manure application is an effective strategy to enhance soil microbial biomass and soil N availability and has the potential to reduce the dependence upon chemical N fertilization.