The fast and effective generation of a concentration gradient by mixing in the microchannel is important for many microfluidic applications. The active control of gradient is useful for applying the measurement of cell responses by dynamic change of environment. The main purpose of this paper is the generation of temporally stable concentration gradient actively. For this purpose, the wave-like pattern of the liquid–liquid interface is produced in the microchannel. In this technique, the high frequency of the wave-like pattern is necessary for reducing the length of the mixing path. High frequency of the wave-like pattern is achieved by employing the newly developed microvalve using tailor-made multilayer piezoelectric actuators (TAMPA) that is compact yet produces large displacements and forces. This paper first details the concept for the concentration gradient generation method. Next, a microvalve (20 × 15 × 15 mm) was designed and produced using TAMPA (8.5 × 10 × 10 mm). Finally, a concentration gradient in two-layered flow was generated with the microvalve. As a result, the generation of a concentration gradient in two-layered flow with active mixing was achieved. Furthermore, it is shown that the concentration gradient can be controlled actively by adjusting the input voltage to TAMPA.