•Synthesis of C-3-O-labeled BODIPY–estradiol conjugates.•Labeling via C4–C8-long alkyl linkers based on CuAAC reaction or etherification.•Molecular docking calculations on two binding sites of human ERα.•Retained estrogenic activity, direct interaction and activation of ERα.•Induction of cellular fluorescence and co-localisation with ERα. Novel BODIPY–estradiol conjugates have been synthesized by selecting position C-3-O for labeling. The conjugation strategy was based on Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) or etherification. Estradiol derivatives used as azide partners bearing an ω-azidoalkyl function through C4–C8-long linkers have been prepared. CuAAC reactions of estradiol azides with BODIPY alkyne furnished fluorescent 3-O-labeled conjugates bearing the triazole ring as a coupling moiety. Williamson etherifications of 3-O-(ω-bromoalkyl)-17β-estradiol derivatives with BODIPY-OH resulted in labeled conjugates connected with an ether moiety. Interactions of the conjugates with estrogen receptor (ER) were investigated using molecular docking calculations in comparison with estradiol. The conjugates occupied both the classical and alternative binding sites on human ERα, with slightly lower binding affinity to references estradiol and diethystilbestrol. All compounds have displayed reasonable estrogenic activity. They increased the proliferation of ER-positive breast cancer cell line MCF7 contrary to ER-negative SKBR-3 cell line. The most potent compound 13a induced the transcriptional activity of ER in dose-dependent manner in dual luciferase recombinant reporter model and increased progesterone receptor's expression, proving the retained estrogenic activity. The fluorescence of candidate compound 13a co-localised with the ERα. The newly synthesized labeled compounds might serve as good starting point for further development of fluorescent probes for modern biological applications. In addition to studying steroid uptake and transport in cells, e.g. in the processes of biodegradation of estrogen-hormones micropollutants, they could also be utilized in examination of estrogen-binding proteins. Display omitted