Luminescent lanthanide complexes that can be covalently bound to proteins have shown great utility as biolabels for highly sensitive time-gated luminescence bioassays in clinical diagnostics and biotechnology discoveries. In this work, three new tetradentate β-diketonate–europium complexes that can be covalently bound to proteins to display strong and long-lived Eu³⁺ luminescence, 1,2-bis[4′-(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-benzyl]-4-chlorosulfobenzene-Eu³⁺ (BHHBCB-Eu³⁺), 1,2-bis[4′-(1″,1″,1″,2″,2″-pentafluoro-3″,5″-pentanedion-5″-yl)-benzyl]-4-chlorosulfobenzene-Eu³⁺ (BPPBCB-Eu³⁺), and 1,2-bis[4′-(1″,1″,1″-trifluoro-2″,4″-butanedion-4″-yl)-benzyl]-4-chlorosulfobenzene-Eu³⁺ (BTBBCB-Eu³⁺ ), have been designed and synthesized as biolabels for time-gated luminescence bioassay applications. The luminescence spectroscopy characterizations of the aqueous solutions of three complex-bound bovine serum albumin reveal that BHHBCB-Eu³⁺ has the strongest luminescence with the largest quantum yield (40%) and longest luminescence lifetime (0.52 ms) among the complexes, which is superior to the other currently available europium biolabels. The BHHBCB-Eu³⁺ -labeled streptavidin was prepared and used for both the time-gated luminescence immunoassay of human prostate specific antigen and the time-gated luminescence microscopy imaging of a pathogenic microorganism Cryptosporidium muris. The results demonstrated the practical utility of the new Eu³⁺ complex-based biolabel for time-gated luminescence bioassay applications.