We propose a new, to the best of our knowledge, rainbow technique called three-dimensional rainbow refractometry (TDRR), with a cylindrical lens in the signal collecting system. With a TDRR model based on the ray transfer matrix developed, it is proved that the tilt angle of the rainbow signal is related to the axial position of the droplet, which helps to obtain the 3D position. By converting rainbow scattering angle calibration into the system parameter calibration, a new rainbow data processing program is written in combination with the model to obtain the refractive index and the particle size. With TDRR, we measured a monodisperse droplet stream of deionized water at room temperature for experimental validation and obtained the refractive index with an absolute error of less than 0.0015, the droplet size with an error within ±5%, and the axial position with an error within ±3%, which demonstrated a high accuracy of TDRR.