Self-assembly techniques have been demonstrated to be a useful approach to developing new functional nanomaterials. In this study, a novel method to fabricate a manganese phosphate self-assembly monolayer (SAM) on a hyperbranched polyester (HBPE-OH) nanoparticle surface is described. First, the second-generation aliphatic HBPE-OH was carboxy-terminated, phosphorylated, and then ionized with manganese by a three-step modification process. The final product of HBPE-AMPA-Mn2+ particles was obtained and characterised by FT-IR spectroscopy, 1H NMR spectroscopy, transmission electron microscopy (TEM), Zeta potential, and energy dispersive spectroscopy (EDS). Moreover, the HBPE-AMPA-Mn2+ particles were used to construct a novel biosensor for detection of superoxide anions (O2•−) released from HeLa cells. Results showed that the response currents of this biosensor were proportional to the O2•− concentration ranging from 0.79 to 16.6 μM, and provided an extremely low detection limit of 0.026 μM (S/N = 3). The results indicate that the particle-decorated electrode surface, which involved a hyperbranched structure and a surface self-assembly technology, proposed here will offer the ideal catalytic system for electrochemical enzymatic sensors.