Dopamine (3,4-dihydroxylphenyl ethylamine) is the most significant neurotransmitter in the human nervous system. Abnormal dopamine levels cause fatal neurological disorders, and thus measuring dopamine level in actual samples is important. Although electrochemical methods have been developed for detecting dopamine with high accuracy, certain substances (e.g., ascorbic acid) in actual samples often interfere with electrochemical dopamine detection. We developed tyrosinase-based dopamine biosensor with high sensitivity and selectivity. An electrochemically pretreated tyrosinase/multi-walled carbon nanotube-modified glassy carbon electrode (tyrosinase/MWNT/GCE) was prepared as an amperometric biosensor for selective dopamine detection. For optimizing the biosensor performance, pH, temperature, and scan rate were investigated. The electrochemically pretreated tyrosinase/MWNT/GCE exhibited not only the highest sensitivity (1,323mAM-1cm-2) compared to previously reported tyrosinase-based dopamine sensors, but also good long-term stability, retaining 90% of initial activity after 30 days. Additionally, ascorbic acid, a major interfering substances, was not oxidized at the potential used to detect dopamine oxidation, and the interfering effect of 4mM ascorbic acid was negligible when monitoring 1mM dopamine. Consequently, the electrochemically pretreated tyrosinase/MWNT/GCE is applicable for highly selective and sensitive dopamine detection in actual samples including interfering substances, thereby extending the practical use to monitor and diagnose neurological disorders.