A one-pot hydrothermal synthesis of three-dimensional (3D), large-area, bimetallic oxide NiCo2O4 (NCO) microflowers has been developed as a novel substrate for surface-enhanced Raman scattering (SERS) detection of flutamide in biological fluids. The 3D flower-like morphology of the NCO is observed via FESEM micrographs, while the orthorhombic phase formation is confirmed through XRD spectra. Due to the presence of multiple coordination cations of the 3D NCO microflowers (such as Ni2+ and Co2+), the high surface area and surface roughness, the NCO-modified indium tin oxide (NCO/ITO) SERS substrate exhibits a linear detection range from 0.5–500 nM with a low limit of detection (LOD) of 0.1 nM. The SERS substrate provides a high enhancement factor of 1.864 × 106 with an accumulation time of 30 s using a laser source of λ = 532 nm, which can be ascribed to the excellent and rapid interaction between the flutamide molecule and the NCO microflower substrate that leads to photoinduced charge transfer (PICT) resonance. The NCO/ITO substrate exhibits excellent homogeneity and high chemical stability. Besides, the substrate displays an excellent selectivity to flutamide molecules in the existence of other metabolites such as urea, ascorbic acid (AA), glucose, NaCl, KCl, CaCl2, and hydroxyflutamide. The NCO/ITO substrate is successful in the trace-level detection of flutamide in simulated blood serum samples. The strategy outlined here presents a novel strategy for the efficacy of transition metal oxides (TMOs) based electrodes useful for a wide variety of bioanalytical applications. Graphical abstract: [Figure not available: see fulltext.]. © 2021, The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.