A series of dinuclear compounds of [Ru(bpy)2(tpphz)Ln(TTA)3](PF6)2 (tpphz = tetrapyrido[3,2-a:2′,3′-c:3′′,2′′-h:3′′′,4′′′-j]phenazine; Ln = Er(III), Nd(III), Yb(III) and Gd(III); TTA = 2-thenoyltrifluoroacetone) have been prepared by attachment of a [Ln(TTA)3] fragment at the vacant diimine site of the luminescent mononuclear complex [Ru(bpy)2(tpphz)](PF6)2. In the solid state, in CH2Cl2 solution and in Tris-HCl buffer solution of these dinuclear complexes Ru–Ln, sensitized near-infrared (NIR) luminescence is observed from Nd and Yb centres following excitation of the d-block unit, which results from the effective Ru → Ln (Ln = Nd, Yb) energy transfer, but no Er-based NIR luminescence is produced. The 3MLCT (MLCT = metal to ligand charge transfer) emission is partly quenched in the Ru–Nd complex, slightly increased in the Ru–Yb complex, and is not changed in the Ru–Er complex. Interestingly, alpha-fetal protein (AFP) tends to decrease the NIR luminescence intensity of the Ru–Nd complex in Tris-HCl buffer solution. A novel NIR luminescent method for the determination of AFP was developed with a linear range of 0.5–18 ng mL−1, and a detection limit of 0.2 ng mL−1 based on 3 times the ratio of the signal-to-noise. Considering the attractive features, such as good selectivity, stability and rapidity, the proposed NIR luminescent method provides promising potential for AFP detection in clinical diagnosis and biomedical applications.