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[en] Highlights: • Design and synthesis of simultaneous Gd3 + and Dy3 + substitutions in β-Ca3(PO4)2 as a potential multifunctional bioprobe. • Both Gd3 + & Dy3 + occupies at Ca2 +(1), Ca2 +(2) & Ca2 +(3) lattice sites of β-Ca3(PO4)2. • Absorption and photoluminescence studies enunciates the dual role of Gd3 + and Dy3 +. • Dual substitutions facilitates excellent computed tomography and magnetic resonance imaging characteristics. A series of Gd3 + and Dy3 + co-substituted β-Ca3(PO4)2 as a multifunctional bio-probe has been developed. Structural analysis revealed preferential occupancy of both Gd3 + and Dy3 + at Ca2 +(1), Ca2 +(2) and Ca2 +(3) sites of β-Ca3(PO4)2 and ~ 4.20 mol.% has been determined as the substitution limit for co-substitutions with individual contribution being ~ 2.20 mol.%. The SAED diffraction pattern of powder samples coincides well with the XRD results. The UV–vis-NIR absorption spectroscopy and photoluminescence of β-Ca3(PO4)2 are emphasized by the dual role of Gd3 + and Dy3 + ions. The dual substitutions exhibited paramagnetic characteristics with Ms of 1.08 emu g− 1 suitable for MRI contrast agents. Cell cytotoxicity assay demonstrates negligible toxicity and hence deliberates its safety in vivo applications. In vitro imaging analysis advocates the potential applicability of the developed multifunctional bio-probe in MRI, CT and optical imaging.