Highly sensitive and selective non-enzymatic glucose detection was developed using nanoporous Ag flowers on a Ni substrate. The cyclic scanning electrodeposition (CSE) method was used to fabricate Ag flowers on a Ni substrate in an alkaline electrolyte. The nanoporous Ag flowers were then formed by repeated CSE in NaOH. The growth mechanisms of the nanoporous Ag flowers were systematically studied, and these mechanisms can be extended to the formation of other metal, bimetal or metal oxide. The synthesized three-dimensional nanoporous Ag flowers on the Ni substrate were used in the electro-oxidation of glucose, demonstrating a wide linear range (0.1 μM to 1 mM), fast response time (<2 s), low detection limit of 0.1 μM (S/N = 3) and a high sensitivity to detect glucose in the presence of uric acid (UA) and ascorbic acid (AA) at the level of their physiological concentrations. Apart from the nanoporous Ag flowers, the formation of a NiO thin layer on the Ni substrate during CSE also contributed to the high selectivity. This work indicates the potential for developing a fast, sensitive, selective and stable electrochemical sensor for diabetes diagnosis.