Biochar could be got from crop straw which contain rich carbon under oxygen free or oxygen limited conditions at low temperature. The application of biochar into soil is beneficial to ease the pressure of handling straw, reduce pollution, reduce greenhouse gas emissions and improve soil quality. This study was carried out in a cornfield containing meadow brown soil at the lower reaches of Liao River which was treated with different amounts of biochar (0, 360, 1 800, 3 600 kg·ha-1) and fertilizer. We investigated the contents of soil available phosphorus (AP), organic P (OP) and total P (TP). We also investigated the enzyme activities of soil acid phosphatase (AcP), alkaline phosphatase (AlP) and phosphodiesterase (PD) via a fluorescence spectroscopy method by using a fluorescent conjugated polymer as the substrate. Soil AP contents increased drastically with the increasing application of biochar, whereas the OP and TP contents exhibited little change. The increase in AP contents was ascribed to the introduction of P into the soil via biochar. Soil AlP and PD activities increased with increasing biochar application. Soil AcP activity increased significantly after the application of the appropriate amount of biochar (1 800 kg·ha-1), whereas it was inhibited by the application of high levels of biochar (3 600 kg·ha-1), perhaps due to the intrinsic alkalinity of biochar. The effect of Biochar inputs on soil phosphorus element and phosphatase activity is the comprehensive embodiment of the soil physical properties, chemical properties, and microbial community structure and metabolic capacity. We should further study such item. The fluorescent microplate method used in this study has many advantages, such as accuracy, rapidness and simple to perform.