Nickel (Ni) is a required element but may become toxic to plants, animals and humans if normal levels are exceeded. To assess the health and environmental effects of Ni, the bioavailable, not total, concentration of Ni in soil must be accurately measured. Urease has an absolute requirement for Ni to function, and this was used to develop a bioassay to assess Ni bioavailability in soil. Bacteria with urease activity were enriched from Spinks sandy soil by growing the cells in Ni-deficient Luria Broth medium. This created a culture with high amount of potential urease activity but with low actual activity because of Ni limitations. The bacterial culture was inoculated into a test soil sample composed of a mixture of acid-washed Spinks sandy soil as a carrier soil and a soil with unknown Ni bioavailability. After four hours of equilibration, the urease response to the bioavailable Ni in the unknown soil was then measured using steam distillation. Results showed that urease activity was positively correlated to Ni bioavailability. The urease bioassay was able to detect as little as 1.99 mg/kg of bioavailable Ni in soil. The coefficient of variation for the urease bioassay was approximately 10%, indicating good precision. A significant correlation (R2 = 0.9945) was observed between the values for Ni bioavailability measured using the urease bioassay method and Mehlich III extraction. This indicates the bioassay method provided a good indication of the bioavailable Ni content in soil samples compared to chemical extraction. Results also showed that a change in soil total C content from 0.009% to 12.7% decreased Ni bioavailability by 50%, and a change in soil pH from 4.15 to 9.94 decreased Ni bioavailability by 70%. The urease bioassay to assess Ni bioavailability in soil is accurate, simple, and sensitive.