The growing need to prevent pathogen outbreaks is irrefutable in the case of the food industry. Early detection in products, especially beverages, contaminated with bacterial strains is vital to avoid infected foods from reaching the consumer. If E. coli is pesent in such foods, it can cause infections. It can also be an indicator of the existence of other harmful coliforms. In this study, we have investigated the detection of Escherichia coli ( E. coli) in orange juice using a portable nanofiber-light addressable potentiometric sensor (NF-LAPS). We have chosen electrospun pH-sensitive poly(vinyl alcohol)/poly(acrylic acid) (PVA/PAA) hydrogel NFs as the sensitive layer. The successful detection of E. coli was reported with the NF-LAPS in less than 1 h. The limit of detection (LOD) measured in the sensor is found to be102 CFU/mL. We have confirmed the selectivity of the biosensor toward E. coli by examining the response of the NF-LAPS against Salmonella typhimurium ( S. typhi), also commonly found in orange juice. Despite the complex nature of orange juice, the response of the biosensor is in no way affected while orange juice is tested as is.