Extremophilic microorganisms are promising candidates for industrial and analytical biocatalysis. Haloferax volcanii, a halophilic archaeon that prefers glycerol over glucose, channels this substrate into central metabolism through glycerol kinase (GK). Here, we report the biochemical properties of H. volcanii GK and its potential for biotechnological applications. An N-terminal His-tagged GK was functional in vivo and yielded 3 mg/L culture-4.5 times more enzyme than a C-terminal StrepII-tagged version. Size exclusion chromatography revealed a glycerol-induced oligomeric shift from homodimer to a dimer-dominant state with detectable tetramer. The purified enzyme showed robust activity across broad pH and salinity ranges, with optimal activity at 100 mM NaCl and 50°C-60°C. It retained catalytic activity in 5%-10% dimethyl sulfoxide (DMSO) and crude glycerol containing methanol. His-GK was freeze-thaw stable and thermotolerant in 2 M NaCl buffers. In the absence of ligands, the enzyme's melting temperature (T(m)) was 80°C. Glycerol increased the T(m) to 85°C, and combinations with MgCl₂ (84°C) or ATP (88°C) provided further stabilization. The highest T(m) (89°C) occurred with all three ligands, suggesting a cumulative stabilizing effect. Kinetic analyses revealed positive cooperativity for glycerol, ATP, and Mg²(+); Mn²(+) and Co²(+) also supported the activity. H. volcanii GK is the first known GK to exhibit positive cooperativity with glycerol and ATP. Its high stability and substrate flexibility support its use in biodiesel waste valorization, in vitro biocatalysis, and biosensor development-applications demanding robust, specific, and stable enzymes.IMPORTANCEThis study reveals that H. volcanii GK exhibits positive cooperativity for glycerol, ATP, and Mg²(+), a kinetic feature not previously reported for glycerol kinases. This behavior enables steep, switch-like responses to small substrate changes, offering unique advantages for biosensor design. Importantly, H. volcanii GK also maintains high activity under extreme salinity, temperature, broad pH, and solvent conditions that typically limit enzyme use in industrial and environmental applications. These traits make this GK an ideal candidate for enzyme-based biosensors, which often suffer from poor tolerance to pH, solvent, and thermal stress. Its robustness supports its use in cross-linked enzyme crystals, an immobilization method that enhances enzyme stability and reusability under harsh conditions. Moreover, GKs are already employed in Mg²(+) detection kits; however, H. volcanii GK's ability to tolerate and respond to diverse divalent cations (e.g., Co²(+), Mn²(+)) broadens their potential for pollutant detection and environmental monitoring. These features collectively position H. volcanii GK as a valuable biocatalyst for biosensing, in vitro diagnostics, and biotechnological applications requiring both precision and durability.