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We investigated the influence of morphology of Ni microstructures modified with Au and Pt on their cell biocompatibility and electrocatalytic activity toward non-enzymatic glucose detection. Synthesis and modification were carried out using a simple and inexpensive approach based on the method of laser-induced deposition of metal microstructures from a solution on the surface of various dielectrics. Morphological analysis of the fabricated materials demonstrated that the surface of the Ni electrode has a hierarchical structure with large-scale 10 μm pores and small-scale 10 nm irregularities. In turn, the Ni-Pt surface has large-scale cavities, small-scale pores (1–1.5 μm), and a few tens of nanometer particles opposite to Ni-Au that reveals no obvious hierarchical structure. These observations were supported by impedance spectroscopy confirming the hierarchy of the surface topography of Ni and Ni-Pt structures. We tested the biocompatibility of the fabricated Ni-based electrodes with the HeLa cells. It was shown that the Ni-Au electrode has a much better cell adhesion than Ni-Pt with a more complex morphology. On the contrary, porous Ni and Ni-Pt electrodes with a more developed surface area than that of Ni-Au have better catalytic performance toward enzymeless glucose sensing, revealing greater sensitivity, selectivity, and stability. In this regard, modification of Ni with Pt led to the most prominent results providing rather good glucose detection limits (0.14 and 0.19 μA) and linear ranges (10–300 and 300–1500 μA) as well as the highest sensitivities of 18,570 and 2929 μA mM–1 cm–2. We also proposed some ideas to clarify the observed behavior and explain the influence of morphology of the fabricated electrodes on their electrocatalytic activity and biocompatibility.