Multi-functional florescent dyes capable of acting as molecular probes in living systems under two-photon microscopy, as well as super-resolution nanoscopy, are of great interest. Herein, a novel donor–π–acceptor (D–π–A) type pyrimidine imidazole derivative with a flexible alkyl chain molecule (EX-1) has been efficiently synthesized through improved Knoevenagel condensation with high yield and relatively large 2PA cross-sections. Furthermore, a cytotoxicity assay and photo-resistance evaluation indicated that such small organic dyes could not only effectively target the endoplasmic reticulum in living cells, but also reveal its ultrastructure under stimulated emission depletion (STED) nanoscopy.