Soil moisture level is crucial to soil phosphorus (P) availability; however, there is no quantitative research on the relation between soil P availability and moisture level. In addition, biochar application could also alter soil P availability at different moisture levels. In this study, a 16 d soil incubation experiment was conducted at a laboratory scale to analyze the effects of soil moisture and P fertilization regimes (P-laden biochar fertilizer and conventional mineral P fertilizer) on soil P availability and fractionation. The results showed that soil P availability was positively correlated with soil moisture level (Pearson coefficients ranged from 0.46 to 0.91). High moisture level would lead to less amount of P in readily available fractions under P-laden biochar application. However, even with less P in readily available fractions, P-laden biochar could maintain soil P availability (117.7 mg P·m⁻²) at a similar level as the conventional P fertilizer (116.1 mg P·m⁻²).