A simple microfluidic concentration gradient generator (MCG) was developed to create concentration gradients on a centimeter scale with a wide range of flow rates. Four groups of inlets (sample and buffer) were successively connected to the main channel to determine the location of inlets and the type of laminar flow and to create the concentration gradients. Nine micromixing channels were derived from the main channel to efficiently mix laminar flow. A micromixing channel whose mixing index was higher than that of a conventional serpentine mixing channel was designed by combining a square wave structure and microchannel sidewall grooves. A spindle-shaped chamber was set at the end of the mixing channel to observe the gradients. The computational fluid dynamic software Fluent was used to predict concentrations by calculating the average mass fraction at the cross section of each outlet. Theoretical simulations indicated that linear concentration gradients would be achieved within seconds in accordance with the fast micromixing principle at a flow rate of 2280 mu L/min, and the gradient shape was steady regardless of the flow rate, which was affected by the location of inlet ports. The uniformity of the mass fraction achieved by the improved mixing channel was better than that of conventional serpentine micromixing channel. The theoretical simulations were verified through the generation of a concentration gradient of methylene blue water solution. Multiple concentrations of an antibacterial drug (ceftizoxime) generated by this MCG were utilized for an antimicrobial susceptibility test using Escherichia coli. Results revealed that the MCG showed potential for concentration gradient-related biochemical study.