In the present study, thermohydraulic performance and entropy generation analysis through a single rectangular microchannel is investigated systematically by numerical simulations. The simulations are carried out by considering three-dimensional thin-wall approximation and validated with the present experimental results and some empirical correlations. The experiment is conducted for stainless-steel rectangular microchannel of width (W)=0.35 mm, height (H)=0.75 mm and length (L)=50 mm for the laminar flow i.e., Reynolds number (Re) in the range of 150–1050 with DI-water as testing fluid. Further extending the numerical simulations, both thermohydraulic performance and entropy generation have been studied by varying the fluid flow and structural parameters of the microchannel. The results demonstrate that the variation of flow pressure and Nusselt number ( Nu) along the channel length agrees with the conventional flow theory. The friction factor value depends upon the aspect ratio (AR) and independent of channel hydraulic diameter (D_h), and channel having an AR value of 1 exhibits the lowest friction factor. The average Nusselt number (〖Nu〗_avg) depends upon both AR and D_h, and the channel having an AR value of more than 1 exhibits higher 〖Nu〗_avg because of higher surface area. The channel D_h and flow Re have significant effect on the total entropy generation (S_G), while AR hasn’t had that much impact because by varying AR from 0.18 to 5.5, the S_G increases by 5% only. Finally, the multi-objective optimization is carried out by using response surface methodology (RSM) to obtain the optimal design parameter condition i.e., W=1 mm, H=0.63 mm, and Re=1050, which fulfills the required objective with 0.8254 desirability.