The seed layer plays a crucial role in achieving high electrical conductivity and ensuring higher performance of devices. In this study, we report fabrication of a solution-gated field-effect transistor (FET) sensor based on zinc oxide nanorods (ZnO NRs) modified iron oxide nanoparticles (α-FeO NPs) grown on a highly conductive sandwich-like seed layer (ZnO seed layer/Ag nanowires/ZnO seed layer). The sandwich-like seed layer and ZnO NRs modification with α-FeO NPs provide excellent conductivity and prevent possible ZnO NRs surface damage from low pH enzyme immobilization, respectively. The highly conductive solution-gated FET sensor employed the calmodulin (CaM) immobilization on the surface of α-FeO-ZnO NRs for selective detection of calcium ions (Ca). The solution-gated FET sensor exhibited a substantial change in conductance upon introduction of different concentrations of Ca and showed high sensitivity (416.8 μA cm mM) and wide linear range (0.01-3.0 mM). In addition, the total Ca concentration in water and serum samples was also measured. Compared to the analytically obtained data, our sensor was found to measure Ca in the water and serum samples accurately, suggesting a potential alternative for Ca determination in water and serum samples, specifically used for drinking/irrigation and clinical analysis.