Novel flower-like silver particles with triangular plates as building block along with functionalized graphene (straggled sheets) and enzymes horseradish peroxidase (HRP) or cholesterol oxidase (ChOx), were obtained on graphite electrode by galvanostatic electrodeposition method. The morphology of the electrodeposits has been characterized using scanning electron microscopy and energy-dispersive analysis of X-ray. The resulting biosensors named Nf/(HRPf - graphene-Ag)/Gr and Nf/(ChOx-f-graphene-Ag)/Gr were evaluated for electrochemical activity using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and chronoamperometry. Optimization of the interdependent experimental parameters such as pH and temperature were achieved and maintained constant throughout the experiments. An activation energy of 2.5 kJ mol⁻¹ was obtained for Nf/(HRP-f-graphene-Ag)/Gr electrode while Nf/(ChOx-fgraphene- Ag)/Gr showed an activation energy of 2.06 and 3.12 kJ mol⁻¹. Furthermore, the former electrode demonstrated a good linear range of 25 μMto 19.35mMwith rapid response time of 3 s and detection limit of 5 μM for hydrogen peroxide. Similarly, the Nf/(ChOx-f-graphene-Ag)/Gr electrode revealed a linear range of 0.1–4.5 mM with rapid response time of 3 s and an excellent detection limit of 0.514 mM for cholesterol. Besides this, the Nf/(HRP-f-graphene-Ag)/Gr and Nf (ChOx-fgraphene- Ag)/Gr electrodes displayed a Michaelis–Menten constant of 0.26 and 0.57 mM, respectively, suggesting high affinity and enzymatic activity. The enhanced performance of biosensors towards detection of substrate and rejection of interferents, provided an evidence for its high antiinterference ability. Additionally the biosensors exhibit long term storage stability and reproducibility with antifouling properties.