There is a strong requirement for label-free, selective and rapidly detection of Protamine (PRTM) due to that the PRTM and Heparin (Hep) have been widely used in clinical treatment. Here, an optical fiber interferometer biosensor that functionalized by layer-by-layer electrostatic assembled chitosan/heparin (CS/Hep) is proposed for PRTM detection. Experimental results proved that the refractive index (RI) sensitivity is related to thickness of CS/Hep overlayer, which is a key factor of sensor's performance. The CS/Hep polyelectrolyte film coating extend its evanescent filed and can also be used as the signal amplification medium for PRTM detection due to the electrostatic interactions between PRTM and Hep. The optimal sensor that coated with 10 bilayers of CS/Hep, demonstrates RI sensitivity of 435.6 nm/RIU as well as ideal stability, and was chosen for PRTM detection. The CS/Hep based biosensor demonstrates a detection limit of 0.48 μg/mL and sensitivity of 0.1238 nm/(μg/mL) in the linear fitting range. Furthermore, the sensor illustrated ideal selectivity in serum samples and well reusability. Overall, the sensor features with high mechanical strength, ease of fabrication, real-time and label-free. This proposed sensor has potential applications for detecting PRTM in clinical applications. © 2023 Elsevier B.V.