The urease enzyme (Urs) was successfully incorporated into a polypyrrole film (PPy) in one simple electropolymerisation step. The films were formed using different dopant anions. The polypyrrole-urease-chloride film (PPy-Urs-Cl) was deposited from a simple chloride dopant, and the polypyrrole-urease-sulfonated-β-cyclodextrin (PPy-Urs-SCD) was formed using a sulfonated-β-cyclodextrin (SCD) dopant. The presence of the Urs within the polymer film was evident from the fibrous morphology, observed in the SEM micrographs, and the presence of nickel, arising from the active site of the urease enzyme. The sensing ability of the films and their enzyme-free counterparts (i.e., PPy-Cl and PPy-SCD) towards urea was investigated. The dopant anion plays an important role in the sensitivity of the polymer films towards urea. The PPy-Urs-SCD film has a superior sensitivity of 5.79 μC μM−1 compared to 0.76 μC μM−1 for the PPy-Urs-Cl polymer film. Furthermore, the negative groups on the SCD eliminate interference from common interfering compounds, such as ascorbic acid.