The demand for rapid and accurate detection methods for Salmonella Enteritidis necessitates the development of highly sensitive and specific biosensors to ensure proper monitoring of food safety and quality requirements in the food sector and to secure human health. This study focused on development of a polyaniline/zinc oxide (PANI/ZnO) nanocomposite film on a gold electrode conductometric immunosensor for detection of Salmonella Enteritidis. The sensor was modified with monoclonal anti-Salmonella Enteritidis antibodies as biorecognition elements. The fabricated sensor was able to detect and quantify the target pathogen within 30 min and showed a good detection range from 101 to 105 colony-forming units (CFU)/mL for Salmonella Enteritidis and a minimum detection limit of 6.44 CFU/mL in 0.1% peptone water. Additionally, the fabricated sensor showed good selectivity and detection limit toward the target bacterium and successfully determined Salmonella Enteritidis content in ultrahigh heat-treated skim milk samples without pretreatment of the food sample.