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Luminescent nanospheres (EuPHS, dav=45 nm) containing 40%(w) of Eu(tta)3dpbt (tta=thenoyltrifluoroacetonato;dpbt=2-(N,N-diethylanilin-4-yl)-4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine) were prepared by encapsulating Eu(tta)3dpbt in a hybrid matrix formed in situ from poly(styrene-co-methyl methacrylate), octyltrimethoxysilane, and poly(octylsiloxane). The EuPHS are promising luminescent markers for bioanalysis because of their good dispersibility in aqueous solutions, high photostability, low cytotoxicity, and bright Eu3+luminescence under excitation at long wavelengths. EuPHS exhibited excel ent visible light-sensitized and near-infrared two-photon-sensitized Eu3+ luminescence properties, with a visible-light excitation peak at 415 nm and an excitation window extending up to 475 nm. The quantum yield for Eu3+ luminescence was 0.31(λex=415 nm, T=23°C), and the two-photon excitation (TPE) action cross section was 5.0×105 GM (1 GM=10-50 cm4·s·photon-1·particle-1) at 830 nm. Bionanoprobes preparedby adsorbing transferrin on the surfaces of EuPHS were successful y applied in target-specific labeling and two-photon-excitation imaging of live HeLa cel s.