For the first time, a novel EpCAM aptamer (SYL3C)-DIBO-AF594 fluorescent conjugate was synthesised by bioorthogonal chemistry utilizing a strain promoted alkyne–azide cycloaddition (copper free click) reaction (SPAAC). The ligation efficiency of SPAAC was improved by freeze–thaw cycles. The obtained conjugate showed target specific binding and aided in the imaging of various EpCAM positive cancer cell lines like MCF7, MDAMB453, Weri-RB1 and PC3.