The generation of concentration gradients is an essential part of a wide range of laboratory settings. However, the task usually requires tedious and repetitive steps and it is difficult to generate concentration gradients at once. Here, we present a microfluidic device that easily generates a concentration gradient by means of push-button actuated pumping units. The device is designed to generate six concentrations with a linear gradient between two different sample solutions. The microfluidic concentration gradient generator we report here does not require external pumps because changes in the pressure of the fluidic channel induced by finger actuation generate a constant volume of fluid, and the design of the generator is compatible with the commonly used 96-well microplate. Generation of a concentration gradient by the finger-actuated microfluidic device was consistent with that of the manual pipetting method. In addition, the amount of fluid dispensed from each outlet was constant when the button was pressed, and the volume of fluid increased linearly with respect to the number of pushing times. Coefficient of variation (CV) was between 0.796% and 13.539%, and the error was between 0.111% and 19.147%. The design of the microfluidic network, as well as the amount of fluid dispensed from each outlet at a single finger actuation, can be adjusted to the user’s demand. To prove the applicability of the concentration gradient generator, an enzyme assay was performed using alkaline phosphatase (ALP) and para-nitrophenyl phosphate (pNPP). We generated a linear concentration gradient of the pNPP substrate, and the enzyme kinetics of ALP was studied by examining the initial reaction rate between ALP and pNPP. Then, a Hanes–Woolf plot of the various concentration of ALP was drawn and the Vmax and Km value were calculated.