The precise and rapid detection of hazardous molecules, microorganisms, pollutants, and toxins currently remains a global challenge. Aflatoxin B1 (AFB1) is a toxic and dangerous product of fungi that considered as cancerogenic, mutagenic, and immunosuppressive for humans and animals. Therefore, the screening of AFB1 in food and beverages plays an important role in preventing foodborne illnesses. In this study, AFB1 molecules were detected in a microfluidic device with integrated polyacrylonitrile/zinc oxide (PAN/ZnO) nanofibers fabricated via a combination of the electrospinning, and atomic layer deposition (ALD) techniques. The structural and optical analyses of PAN/ZnO nanofibers were performed and samples with the most suitable properties were utilized for AFB1 detection. In order to obtain the biorecognition layer toward AFB1, PAN/ZnO samples were modified by (3-Aminopropyl)triethoxysilane (APTES), and glutaraldehyde (GA), bovine serum albumin (BSA) and monoclonal antibodies (Anti-AFB1). Subsequently, photoluminescence (PL)-based immunosensor was integrated into a microfluidic cell and tested for AFB1 detection. The mechanism of PL changes caused by AFB1 & Anti-AFB1 complex formation was analyzed and developed. The proposed approach enables the detection of AFB1 with a lowest concentration (LOD) of about 39 pg/ml, while the sensitivity range was evaluated as 0.1-20 ng/ml. The obtained values of LOD and sensitivity, as well as the simplicity of the detection method, make this approach a prospect for the further application.