Poly(lactic acid) (PLA) and poly(ε-caprolactone) (PCL) are two immiscible polymers which are biodegradable and bioabsorbable, being therefore widely applied for medical materials. The formation of homogeneous fibers of PCL by electrospinning represents a difficult task due to the low viscosity of the solutions of this polymer in comparison to the PLA solutions. In addition, the development of simple techniques for the characterization of these systems represents a field of interest. In this paper, nanofibers of PLA/PCL (1:1 w/w) in a CHCl 3 :MeOH (3:1 w/w) solvent system with and without 4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI), a perichromic dye, were electrospun. The electrospun nanofibers obtained present a homogeneous diameter distribution, with average of 874 ± 205 nm in optimization condition for PLA/PCL and 627 ± 199 nm for PLA/PCL/DSMI. The analysis of the differences in the fluorescence emission of DSMI distributed in each polymer of the nanofibers allowed the direct investigation of the polymeric domains. It is proposed that these emission differences result from the preferential stabilization of resonant forms of the dye with each polymeric chain. For the PLA−DSMI system, green emission was observed, while for PCL−DSMI, red emission occurred. The fluorescence results corroborated thermal analyses, where the absence of solvents was possible to confirm. The nanofibers were compared with the dye anchored in an xerogel, which did not exhibit different emission regions due to its homogeneous distribution in the matrix.