Physiological amino acids (AAs) are important indexes for monitoring various diseases, such as cancers. This study proposes a polymer-based separation method in the presence of mixed micelles for the determination of AAs by capillary electrophoresis with light-emitting diode-induced fluorescence. The separation of 18 amino acid-cyano[f]benzoisoindoles (AA-CBIs) was successfully achieved using a solution of polyvinylpyrrolidone (PVP, 5% w/v, Mavg 1,300,000 Da). Moreover, we also demonstrated that the mixed micelles composed of sodium dodecyl sulfate and isopropanol may change the migration order of the AA-CBIs and greatly improve the speed of the separation. With the exception of proline, 21 AA-CBIs from plasma, including high isoelectric point AAs (lysine, ornithine and arginine), were identified under the optimized separation conditions with minimal matrix effects from the plasma. The results of this study demonstrated distinct advantages of the proposed method, such as simplicity, high efficiency and cost-effectiveness, which provide great potential for the diagnosis of several important diseases, including carcinomas, aminoacidopathies and neurotransmission disorders.