Microfluidic channel systems can be used for various biomedical applications, including drug administration, wound healing, cell culture research, and many others. A 3D microfluidic channel system has enormous potential over conventional microfluidic channel systems, including the capacity to simulate biological events in a laboratory setting. This system has the ability to recreate biological phenomena such as concentration gradient generators (CGGs). Microfluidic CGGs have complex fabrication when built into a 3D channel system. These complex systems can be built with complicated processes such as plasma bonding, which requires expensive setup and fine equipment. Therefore, in this study, a smart additive manufacturing technique is applied for an enormous review of the design and fabrication process, which is optimized for different operating conditions. This study employs a 3D printed removable channel mold to avoid the complex fabrication technique of microfluidic channels, allowing the direct casting of polydimethylsiloxane without extra bonding stages. The proposed design comprises dual mixing stages, incorporating a 3D mixer configuration and a converging output to attain the desired gradient outcome. Optimization is performed to achieve the best operating conditions by using response surface methodology, with channel dimension