Two different fluorescent block copolymers of poly(lactic acid) (PLA) and polyethylene glycol (PEG) containing fluorescein grafted to the polymer chain were synthesized by Ugi four-component condensation (UFCC). The structure of these PEG–PLA copolymers was confirmed by ¹H-NMR and fluorescence spectroscopy. Paclitaxel-loaded fluorescent microspheres (PCT-FMS1 and PCT-FMS2) were prepared from them by the single-emulsion solvent evaporation method. A kinetic study of drug release in vitro using high-performance liquid chromatography showed a prolonged and controlled release of paclitaxel. Anticancer activity of release medium against colorectal cancer cell line (Caco-2) was determined using the cell viability assay. Paclitaxel-loaded microspheres were able to inhibit cancer cell growth and colony formation. The main contribution of this work is to propose a new application for UFCC in the preparation of biomasked fluorescent drug delivery systems able to improve cancer treatment.