【Objective】Different phosphorus（P）fertilizer rates were applied to clarify the effect of P fertilizer on proline biosynthesis and accumulation and drought-resistance in sugarcane.【Method】Sugarcane variety YT-55 was used as the plant material for barrel experiment. Under the normal and drought conditions, four P levels were designed. The physiological and biochemical indexes such as the expressions of P5CS and δ-OAT genes, enzyme activities and the contents of free proline and chlorophyll were determined.【Result and conclusion】The expressions of P5CS and δ-OAT genes,enzyme activities and the free proline content were affected by the ratios of N, P and K fertilizers under the normal and drought conditions. When the P level was 900 kg·hm^-2, both P5CS and δ-OAT activities were relatively low, and the free proline content was the lowest in the plants under the normal water condition. The P5CS activity was the highest; the δ-OAT activity was also at a high level; and the free proline content was up to the maximum amount under drought stress. Based on the results, the optimum levels of N, P and K fertilizers are 918 kg·hm^-2 urea, 900 kg·hm^-2 calcium superphosphate and 750 kg·hm^-2 potassium chloride in Zhanjiang lateritic soil. The results of correlation, partial correlation and path analysis indicated that the P5CS was more effective than δ-OAT in free proline biosynthesis and accumulation of sugarcane under drought stress. The main pathway of free proline biosynthesis and accumulation is glutamic acid pathway (Glu→Pro) supplemented by ornithine pathway (Orn→ Pro) under drought stress.