In this study, we report on the three-dimensional (3D) characterization of a spray in terms of its droplet Sauter mean diameter (SMD) using the laser-induced fluorescence (LIF)/Mie ratio technique. The spray structure is analyzed for a multi-hole direct-injection spark ignition (DISI) injector. A calibration curve to convert the LIF/Mie ratio to droplet diameter is deduced using LIF/Mie imaging and analysis of single droplets generated by a droplet generator. The DISI spray investigated here is optically sectioned by means of two-phase structured laser illumination planar imaging to suppress the intensity of multiple light scattering from LIF and Mie images prior to their ratio. A series of calibrated LIF/Mie ratio images of spray is then recorded at several depths along the direction following the light sheet scanning of the spray. The droplet SMD ranges from less than 5 μm up to a maximum of 50 μm in single-shot images. The averaged SMD results (1–30 μm) obtained by using the calibration curve from the droplet generator are compared with measurement results from phase-Doppler anemometry. Finally, a 3D map is reconstructed from the successive 2D layers generated from spray scanning. The resulting 3D representation of the droplet SMD shows a non-symmetric spray structure produced by the studied multi-hole injector, which cannot be resolved by analyzing only one central plane.