In order to study the effect of phosphorus solubilizing fungal flora on phosphorus availability in soil, firstly, the combined effects of three different kinds of phosphorus solubilizing fungi (1 strain belonged to Mortierella Z1, 1 strain belonged to Penicillium Z2 and 1 strain belonged to Aspergillus niger Z3) were studied in the laboratory, and then the best strain combination Z1+Z2+Z3 was determined, and Z1, Z2 and Z3 flora were selected as test strains in the experiment. Then, the potted rapeseed experiment was conducted to study the effect of phosphorus solubilizing fungi combined with insoluble phosphorus (tricalcium phosphate and phosphate rock powder) on phosphorus availability in soil. The results showed that the contents of soil available phosphorus, organic matter, alkaline phosphatase, invertase and rapeseed yield of phosphorus solublilizing fungi treatment significantly increased by 60.00%, 20.21%, 56.45%, 53.81% and 14.38%, respectively, compared with substrate treatment. The above indicators of the treatment of phosphorus solubilizing fungi combined with insoluble phosphorus were higher than those of single application of phosphorus solubilizing fungi. There was no difference in the soil maximum phosphorus uptake between phosphorus solubilizing fungi treatment and substrate treatment. Phosphorus solubilizing fungi combined with insoluble soluble phosphorus could significantly reduce the soil maximum phosphorus uptake. The maximum phosphorus uptake of phosphorus solubilizing fungi + tricalcium phosphate treatment and phosphorus solubilizing fungi + phosphate rock powder treatment significantly decreased by 158.7 and 47.6 mg/kg compared with phosphorus solubilizing fungi treatment. Soil adsorption constants of the phosphorus solubilizing fungi were lower than those of corresponding substrate treatments. Phosphorus solubilizing fungi could reduce phosphorus adsorption by soil. The results showed that the phosphorus solubilizing fungi should be combined with insoluble phosphorus, which had a positive effect on improving soil phosphorus availability.