Currently, It is vital to develop a material that can be used to detect small concentrations of tumor markers at an early stage of cancer for easy management. Organic dyes and semiconductor quantum dots (QDs) are among the most commonly used detection approaches, but they are limited by either photo-bleaching or innate toxicity. This is evident with QDs, which are composed of heavy metals such as CdSe, CdS, and CdTe. In this condition, free radicals from QDs may be released into the bloodstream, resulting in normal cellular toxicity. In contrast, compared to their conventional counterparts (organic dyes and QDs), lanthanide (Ln³⁺) nanocomposites possess superior physicochemical features, including long-lived luminescence (from several to tens of milliseconds), large antenna-generated stokes or anti-stokes shifts, high resistance to photo-bleaching and photo-blinking, narrow emission bands, and low toxicity. Therefore, Ln³⁺ probes are considered to represent a new generation of luminescent nano-bio-probes for multipurpose biomedical applications, such as in-vitro bio-detection and in-vivo bio-imaging. In this study, two new systems of mixed lanthanide oxide NPs, either terbium/europium oxide NPs, coated with alginate-polydopamine (Alg-PDA), Tb/Eu@AlgPDA and Dy/Er@AlgPDA, were investigated and optimized for Folic acid (FA, as cancer targeting ligand), and PDA, mussel-inspired properties in bio-imaging applications. The Tb/Eu@AlgPDA system outperformed the Dy/Er@AlgPDA system in terms of photo-luminescent efficacy, with more intense and crisp emission peaks on fluorescence intensity. Furthermore, the Tb/Eu@AlgPDA nano-system showed excellent in vivo biocompatibility and intense green fluorescent emission in treated zebrafish embryos, suggesting that Tb/Eu@AlgPDA can potentially be used in fluorescence-based diagnosis. Therefore, these results suggest that Tb/Eu@AlgPDA-DOX NPs can be used as fluorescent imaging probes for accurate cancer diagnosis.