Dopamine (DA) is an important neurotransmitter which indicates the risk of several neurological diseases. The selective determination with low detection limit is necessary for early diagnosis and prevention of neurological diseases associated with abnormal concentration of DA. The purpose of this study is to fabricate a poly(3-aminobenzylamine)/poly(sodium 4-styrenesulfonate) (PABA/PSS) multilayer thin film for use as an electrochemical DA biosensor. The PABA was firstly synthesized using a chemical oxidation method of 3-aminobenzylamine (ABA) monomer with ammonium persulfate (APS) as an oxidant. For electrochemical biosensor, the PABA/PSS thin film was fabricated on fluorine doped tin oxide (FTO)-coated glass substrate using the layer-by-layer (LBL) self-assembly method. The optimized number of bilayers was achieved using SEM and cyclic voltammetry (CV) results. The electroactivity of the optimized LBL thin film toward detection of DA in neutral solution was studied by CV and amperometry. The PABA/PSS thin film showed good sensitivity for DA sensing with sensitivity of 6.922 nA.cm−2.µM−1 and linear range of 0.1–1.0 µM (R2 = 0.9934), with low detection limit of 0.0628 µM, long-term stability and good reproducibility. In addition, the selectivity of the PABA/PSS thin film for detection of DA under the common interferences (i.e., ascorbic acid, uric acid and glucose) was also presented. The prepared PABA/PSS thin film showed the powerful efficiency for future use as DA biosensor in real sample analysis.