The manufacturing of conventional enzymatic biosensors produced via a layer-by-layer (LbL) approach requires expensive instrumentation, and in most cases involves a complex, resource and time-consuming fabrication process. Moreover, LbL assemblies are prone to mechanical instability that leads to irreversible changes in sensor architecture and morphology resulting in degradation of enzymatic activities and insufficient signal reproducibility. Hence, novel fabrication techniques for the production of enzymatic biosensors that are instrumentally controlled and allow reproducible, simultaneous multi-analyte detection with high specificity, temporal and spatial resolution are greatly required. Herein, we report on the development of a novel, fully instrumentally controlled, one-step synthesis approach for the production of nanoparticle-based enzymatic biosensors. The approach relies on a simultaneous encapsulation of the enzyme (glucose and alcohol oxidases), a fluoropolymer (Nafion) and noble metal nanoparticles via co-deposition from a phosphate multiple electrolyte on top of the sensor surface. Remarkably, electrochemical studies revealed that nanoparticle-based biosensors produced by this novel fabrication approach display a significantly enhanced mechanical stability (more than several orders of magnitude higher) without loss of biological activity or leakage of the enzyme or Nafion, and advanced synthesis reproducibility (40 times higher) in comparison to LbL analogues.