In order to provide a scientific basis for optimizing the selection of determination methods for microbial biomass phosphorus (MBP) content in acidic soils, this study conducted a systematic comparison of three methods for determining the content of MBP in south subtropical acidic soils: the chloroform fumigation-sodium bicarbonate extraction method (SB method), the chloroform fumigation-ammonium fluoride-hydrochloric acid extraction method (AFHC method), and the chloroform fumigation-hydrochloric acid-sulfuric acid extraction method (HASA method). The results showed that the spiked recovery rates of inorganic phosphorus for the three methods were 21.62%, 27.39%, and 43.48%, respectively. Acidic soils exhibited strong adsorption and fixation of inorganic phosphorus, and none of the three methods fully extracted inorganic phosphorus from the soil. The phosphorus increment of SB method was significantly lower than that of the other two methods, indicating insufficient phosphorus extraction. The phosphorus increments of AFHC and HASA methods were comparable, with good phosphorus extraction efficiency. The determination results of the three methods showed significant differences (P<0.05). The filtrate color of SB method interfered with detection, with a coefficient of variation (CV) of 17.50%, and its precision was significantly lower than that of the other two methods. AFHC method had a CV of 8.81%, indicating good precision, but turbid filtrates occurred in some acidic soil extracts, affecting normal detection. HASA method had a CV of 8.76%, showing good precision, with clear acidic soil extraction filtrates and minimal detection interference, and this method has obvious detection advantages and can be used for the determination of MBP content in south subtropical acidic soils.