In this study, we assessed the effects of biochar application for five years on soil phosphorus (P) factions by chemical sequential P fractionation, phosphatase activity, and 31P NMR. Four treatments were included in this research: two biochar (0 and 2.63 t ha−1) levels blended with two P fertilizer levels (0 and 26 kg P ha−1), respectively. We found elevated levels of soil pH, total carbon (C), and total P in soil exposed to biochar. The inorganic (Ca8 – P, Al – P, Fe – P, and Ca10 – P) and organic (LOP and HROP) P fractions were all enhanced by biochar regardless of the application of P fertiliser. 31P NMR spectra further showed that ortho – P, monoester – P, and diester – P levels in the soil with added biochar combined with P fertiliser were 464.5 mg kg−1, 74.3 mg kg−1, and 13.9 mg kg−1, respectively, which were the highest among all treatments. Biochar application increased alkaline phosphomonoesterase activity by 24.4–32.7% and decreased phosphodiesterase activity by 10.2–11.0%. In conclusion, biochar application significantly improves soil P fractions and regulates phosphatase activity. [ABSTRACT FROM AUTHOR]