Unlike the ideal conditions considered in most previous studies, the actual cooling flow passage inside gas turbine blades is extremely complex. This complexity is due to the geometrical restrictions resulting from the external cooling holes and blade shape, which change the secondary flow and flow non-uniformity of the internal cooling flow. This study conducted an experimental and numerical analysis to characterize the secondary flow and flow non-uniformity in a realistic internal cooling serpentine passage. Magnetic resonance velocimetry was utilized to measure the average three-dimensional–three-components of the mean velocity. By integrating the flow field, parameters indicating the flow non-uniformity and secondary flow strength were obtained. Reynolds-averaged Navier–Stokes simulations were also conducted, and the Reynolds stress transport model showed relatively good performance when predicting the separation bubble in the U-bend. The secondary flow intensity exponentially decreases after the U-bend, but the rib turbulators maintain the secondary flow at a certain level. Additionally, the high-velocity regions in the inlet zone and beyond the separation bubble create significant flow non-uniformity and inherent shear. At the same time, the turbulence intensity becomes strong at the low-velocity region, which is key for heat transfer enhancement. Therefore, high flow non-uniformity has the potential to enhance heat transfer.