A selective, water-compatible and disposable complex imprinted polymer (CIP) membrane was prepared for L-phenylalanine detection combined with solid phase fluorescence detection. L-Phenylalanine coordinated with aluminum(III) acted as a complex template. The solid phase fluorescent signal was monitored to quantify L-phenylalanine after in situ derivatization with o-phthalaldehyde. Several parameters determining the performance of the CIP membrane were studied and optimized. The resulting CIP membrane exhibited good fluorescent response and selectivity towards L-phenylalanine in aqueous environments. The linear response of the CIP membrane to L-phenylalanine was in the range of 5.0–100 μmol L−1 with a limit of detection of 0.90 μmol L−1. The prepared CIP membrane was successfully applied in the analysis of L-phenylalanine in drinks and human urine with recoveries of 83.5–101.3% and RSDs ranging from 7.2% to 9.5%. The proposed method was compared with a high performance liquid chromatography method. The results showed that the developed method has satisfactory sensitivity, accuracy and precision for the selective determination of L-phenylalanine in water-abundant systems.