A bienzymatic sensing layer containing two enzymes able to work sequentially, choline oxidase (ChOD) and phospholipase D (PLaseD), was used to design an electrochemical biosensor for the detection of either a water-soluble (choline) or insoluble (phosphatidylcholine) substrate. A photocrosslinkable polymer, poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), was used as host-matrix for enzyme immobilization. Controlled amounts of PVA-SbQ and of the two enzymes were directly coated on a platinum disk, then photopolymerized. The compatibility of working conditions for choline and phosphatidylcholine detection in the presence of Triton X-100 and CaCl2was investigated. The effect of the activity ratio PLaseD / ChOD on the sensor performance was determined. The sensitivities to choline and to phosphatidylcholine were 18 mA.1mol−1and 0.66 mA.1.mol−1respectively, the detection limit being 1.5.10−8M for choline and 1.5.10−6M for phosphatidylcholine. The linear range extended up toca. 10−4M for choline andca. 2.10−5M for phosphatidylcholine and the response time was close to 30 seconds for choline andca. 2 min for phosphatidylcholine.