Palladium nanoparticle-bacterial cellulose (PdBC) hybrid nanofibers were synthesized by in-situ chemical reduction method. The obtained PdBC nanofibers were characterized by a series of analytical techniques. The results revealed that Pd nanoparticles were evenly dispersed on the surfaces of BC nanofibers. Then, the as-prepared PdBC nanofibers were mixed with laccase (Lac) and Nafion to obtain mixture suspension, which was further modified on electrode surface to construct novel biosensing platform. Finally, the prepared electrochemical biosensor was employed to detect dopamine. The analysis result was satisfactory, the sensor showed excellent electrocatalysis towards dopamine with high sensitivity (38.4 µA·mM−1), low detection limit (1.26 µM), and wide linear range (5–167 µM). Moreover, the biosensor also showed good repeatability, reproducibility, selectivity and stability and was successfully used in the detection of dopamine in human urine, thus providing a promising method for dopamine analysis in clinical application.