Flexible and sensitive strain sensors can be utilized as wearable sensors and electronic devices in a wide range of applications, such as personal health monitoring, sports performance, and electronic skin. This paper presents the fabrication of a highly flexible and sensitive strain sensor by 3D printing an electrically conductive polydimethylsiloxane (PDMS)/multi-wall carbon nanotube (MWNT) nanocomposite on a PDMS substrate. To maximize the sensor’s gauge factor, the effects of MWNT concentration on the strain sensing function in nanocomposites are evaluated. Critical 3D printing and curing parameters, such as 3D printing nozzle diameter and nanocomposites curing temperature, are explored to achieve the highest piezoresistive response, showing that utilizing a smaller deposition nozzle size and higher curing temperature can result in a higher gauge factor. The optimized 3D printed nanocomposite sensor’s sensitivity is characterized under cyclic tensile loads at different maximum strains and loading rates. A linear piezoresistive response is observed up to 70% strain with an average gauge factor of 12, pointing to the sensor’s potential as a flexible strain sensor. In addition, the sensing function is almost independent of the applied load rate. The fabricated sensors are attached to a glove and used as a wearable sensor by detecting human finger and wrist motion. The results indicate that this 3D printed functional nanocomposite shows promise in a broad range of applications, including wearable and skin mounted sensors.