A convenient procedure for the preparation of the fluorescent dye 4鈥?5鈥?dichloro-2鈥?7鈥?dimethoxy-5(6)-carboxyfluorescein (JOE) is reported; the overall yield achieved starting from isovanillin is 10 times higher (40% vs 4%) compared to the known procedure. Isomers (5- and 6-) are easily chromatographically separable as pentafluorophenyl esters of 3鈥?6鈥?O-bis(cyclohexylcarbonyl) derivatives. Four non-nucleoside JOE phosphoramidites based on 5- and 6-isomers and flexible 6-aminohexanol (AH) or rigid 4-trans-aminocyclohexanol (ACH) linkers have been prepared and used for oligonucleotide labeling. Spectral and photophysical properties of 5鈥?JOE-modified oligonucleotides have been studied. Fluorescence quantum yield of the dye correlates with the nature of the linker (rigid vs flexible) and with the presence of dG nucleosides in close proximity to a JOE residue.