A series of six 2,5-disubstituted 1,3,4-oxadiazole derivatives with various length of conjugation have been synthesized and their optical properties including UV-visible absorption spectra, fluorescence emission spectra, molar absorption co-efficient, Stokes shift and the relative fluorescence quantum yield were measured in a variety of organic solvents. Correlation of the absorption spectra and fluorescence emission response of the 2,5-disubstituted 1,3,4-oxadiazole derivatives with the substituent effect revealed that the optical response can easily be tuned toward red shift by increasing conjugation length. The synthesized compounds were further employed in bioimaging assay in order to investigate their potential as an efficient cell staining agent using L-929 cells under confocal fluorescence microscope which showed scrupulous cell permeability and no toxicity as assessed by MTT assay.