Widespread release of cyanide from industrial activities represents a significant environmental challenge due to its acute toxicity and adverse effects on biological systems. In response to this concern, this study focused on the production of rhodanese from Kocuria rhizophila under submerged fermentation conditions and the assessment of its relevance for cyanide detoxification applications. A soil-derived Gram-positive bacterium was isolated and identified as Kocuria rhizophila based on morphological traits, biochemical profile-based VITEK 2 analysis, and 16S rRNA gene sequencing. Preliminary screening confirmed rhodanese production with an activity of 0.968 RU/mL. Under cyanide-induced submerged fermentation, enzyme production followed a growth-associated pattern and reached maximal activity at 40 h under optimized conditions (35 °C, pH 8.0). Partial purification using sequential precipitation and chromatographic steps enhanced enzyme purity, and SDS–PAGE analysis of the final fraction revealed protein bands at approximately 40, 140, and 260 kDa. Biochemical characterization showed Km values of 33.9 mM for KCN and 19.7 mM for sodium thiosulfate, with a Vmax of ~5.6 µmol min⁻¹ mL⁻¹ for KCN and optimal activity at pH 7–8 and 35 °C. Functional assays demonstrated efficient cyanide detoxification, achieving >85% conversion of KCN, ~92% of NaCN, and 65–77% of Ca (CN)₂ within 60 min in vitro. Collectively, these findings demonstrate that Kocuria rhizophila represents a promising microbial source of rhodanese with efficient cyanide-detoxifying activity, highlighting its potential for biotechnological and environmental remediation applications.