This study discusses an electrochemical biosensor developed for detecting dopamine (DA), utilizing a biosurfactant as its transducer element. Biosurfactants are amphiphilic molecules derived from microorganisms such as fungi, bacteria, and yeast, playing a crucial role in this biosensor. Their hydrophilic and hydrophobic moieties enhance analyte sensitivity, making them ideal for this application. In this work, we precisely fabricated a novel electrochemical biosensor for DA detection by modifying a carbon paste electrode with a biosurfactant (BSB/poly AB25/CPE). We examined the electrochemical behavior of DA across potentials ranging from 0.0 V to 0.8 V, anticipating a possible electrochemical redox mechanism. Additionally, the influence of electrolyte pH on redox behavior was investigated, with pH 7.0 identified as the optimal pH. Key kinetic parameters were determined through scan rate experiments, specifically a charge transfer coefficient of 0.386 and a heterogeneous rate constant of 4.92×10⁻⁵ s⁻¹. The biosensor also demonstrated favorable analytical parameters, including a limit of detection (LOD) of 3.2×10⁻⁷ M and a limit of quantification (LOQ) of 1.06×10⁻⁶ M. The biosensor exhibited excellent repeatability, reproducibility, and stability, confirming its practical utility. Finally, the developed biosensor was successfully applied to quantify DA concentration in pharmaceutical formulations, validating its potential for real-world applications.