In this work, the possibility of using planar droplet sizing (PDS) based on laser-induced fluorescence (LIF) and Mie scattering was investigated within the framework of measuring the droplet Sauter mean diameter (SMD) of direct-injection spark-ignition (DISI) spray systems. For this purpose, LIF and Mie signals of monodisperse fuel droplets produced by a droplet generator were studied at engine relevant diameters (20–50 µm). The surrogate gasoline fuel Toliso (consisting of 65 vol. % isooctane, 35 vol. % toluene) and the biofuel blend E20 (consisting of 80 vol. % Toliso, 20 vol. % ethanol) were used and which were doped with the fluorescence dye “nile red”. The effects of ethanol admixture, dye concentration, laser power, and temperature variation on the LIF/Mie ratio were studied simultaneously at both macroscopic and microscopic scale. The deduced calibration curves of the LIF and Mie signals of both fuels showed volumetric and surface dependent behaviors, respectively, in accordance with the assumptions in the literature. The existence of glare points and morphology-dependent resonances (MDRs) lead to slightly higher LIF and Mie exponents of E20 in comparison to Toliso. In principle, these calibration curves enable the determination of the SMD from LIF/Mie ratio images of transient fuel sprays.