Nickel is an important metal in the adoption of greener technologies needed to transition to cleaner economies, and mines face pressure to source it more sustainably. Nature has evolved nickel-specific ATP-binding cassette (ABC) importers that can transport nickel across a cell’s membrane from the environment into the cytoplasm. Specifically, their solute-binding component known as the nickel-binding protein (NiBP) scavenges the cell’s periplasmic space to deliver metals to the cognate permease complex for transport. Existing methods for metal-binding characterization are not amenable to protein engineering efforts that rely on screening libraries of variants with different substrates. Using natural changes in protein fluorescence during a metal-binding event, we first optimized an assay previously reported in literature to measure the binding affinity (K¬D) of NiBP complexes with nickel and other metals. This assay was validated by determining the KD of the well-characterized NiBP CjNikZ from Campylobacter jejuni, which was comparable to reported values, so it was then used to demonstrate the presence of Ni(II)-binding activity in the uncharacterized NiBP called CcNikZ-II from Clostridium carboxidivorans. Next, we determined the crystal structure of CcNikZ-II, which revealed the potential Ni(II)-binding site located close to a short variable loop. Mutagenesis identified the CcNikZ-II residues involved in Ni(II) binding, but the role of the variable (v-)loop of this protein (TEDKYT) remained unclear. We purified nine CcNikZ-II homologues and determined the nickel-binding affinity of these proteins using an intrinsic fluorescence quenching assay, which showed all these proteins have higher KD for Ni(II) than CcNikZ-II. Furthermore, we replaced the CcNikZ-II v-loop sequence (TEDKYT) with those from the other homologues with higher affinity and found that the engineered CcNikZ-II variants have a higher binding affinity to Ni(II). Metal promiscuity screening further demonstrated the importance of the secondary coordination sphere in controlling affinity and specificity. Finally, an engineered E. coli strain (Ni_v.1) was created and tested in 10 ppm NiCl2 solution matching environmental conditions and demonstrated 7-fold improvement in nickel bioaccumulation performance compared to controls. Thus, both wildtype and engineering microbial NiBPs can be engineered for improved metal binding and selectivity and used for developing bio-based technologies for metal recovery applications.