New carbon materials coordination transition metal as new biocatalytic systems for biosensing are among the desired goals. To reach those purposes, we prepared an oxidase-like active iron nanoparticle encapsulated in boron-nitrogen co-doped carbon nanotubes (Fe/BNC) as a nanozyme, which displayed fascinating oxidase activity and excellent stability. The morphology and structural composition of the as-synthesized Fe/BNC nanocomposites were characterized by scanning transmission electron microscopy and some spectroscopic instruments. Based on this, a new electrochemical sensor was fabricated for the simultaneous determination of DA and UA. Compared with electrode modified by BNC, the modified electrode by Fe/BNC can effectively enhance the electron transport ability of BNC and significantly improve the selectivity and sensitivity of the simultaneous determination of DA and UA. The results show that Fe/BNC can simultaneously detect DA and UA with a peak potential separation of 139 mV. The calibration curves for DA and UA ranged from 1 to 630 and 0.5 to 2065 μM, with detection limits of 0.8 and 0.28 μM, respectively. In addition, the proposed sensor has been successfully applied to the detection of DA and UA in human serum with satisfactory results, suggesting a fascinating potential application in biological analysis.