A novel fluorescent sensing film has been proposed for sensitive determination of Zn2+ ion in EtOH-HEPES buffer solution (20 mM HEPES, 50:50, v/v, pH 7.0). The novel sensor based on incorporating a novel qaudridentate Schiff base N,N’-bis-(1-hydroxypheylimine)2,5-thiophenedicarobxaldehyde (HPTD) as ionophore in the plasticized PVC membrane containing bis(2-ethylhexyl)sebecate (DOS) as plasticizer. The novel optode membrane works on the basis of a cation-exchange mechanism and shows a significant fluorescent emission enhancement on exposure to HEPES buffer solution of pH 7.0 containing Zn2+ ion. Under optimal conditions, the proposed sensing film displays a linear range of 1.0 × 10−12 to 8.6 × 10−4 M with a limit of detection 5.3 × 10−13 M. The response characteristics of the sensor including reversibility, reproducibility, response time and lifetime are discussed in detail. The optode membrane has been applied to determine Zn2+ in various real water samples.