It is important to consider tobacco stalks as sources of biochar and improve the soil quality of tobacco-producing areas in Southwest China. Therefore, in this study, we investigated the effects of biochar produced from tobacco stalks on soil inorganic phosphorus (P) fractions in bulk and rhizosphere soils. We also analyzed the factors influencing these effects in tobacco-planting fields. Biochar was applied to two experimental fields in Heishi and Qianxi in Bijie City, Guizhou Province in China, at application rates of 0, 5, 20, and 40 t ha⁻¹. Subsequently, the soil inorganic P (P_i) fractions, pH, and exchangeable and hydrolytic acidities were analyzed, while the factors influencing the soil P_i fractions were determined by redundancy analysis (RDA). The results indicated that the biochar application rate, experimental site, and tobacco roots all affected the soil P_i fractions. Aluminum-phosphate (Al-P), iron-phosphate (Fe-P), and occluded-phosphate (O-P) levels in the bulk soil had significantly increased with biochar application in the Qianxi site. Meanwhile, there was a reduction in Al-P, Fe-P, and O-P levels in the rhizosphere soil in the Heishi site but not in the Qianxi site. The calcium-phosphate (Ca-P) levels in the soil were also greatly reduced in the Heishi site, while in the Qianxi site, the Ca-P levels were relatively constant in the bulk soil and increased in the rhizosphere soil. Furthermore, the exchangeable and hydrolytic acidities decreased with increasing biochar application. Soil P_i fractions varied according to biochar application rate and experimental site. We found that 20 t ha⁻¹ was an appropriate biochar application rate for enhancing soil P_i fractions. In addition, the soil P_i fractions were negatively correlated with the dissolved organic carbon (DOC) content but were positively correlated with the hydrolytic acidity.