In this work, imidazole- or imidazolium-based benzothiadiazole ligands functionalized on graphene oxide combined with cholesterol oxidase constitute efficient, robust, and easy-to-handle materials with high biosensing activity for the detection of cholesterol by colorimetric methods. The presence of lanthanum(III) supported on graphene oxide as a possible coordinating site for the benzothiadiazole ligands was also evaluated, and its bioactivity was compared to that of the analogous material without the rare-earth metal. Our results demonstrated that graphene oxide functionalized with 4,7-bis-(imidazol-1-yl)-2,1,3-benzothiadiazole exhibited the best performance for the quantification of total cholesterol with a sensitivity of 0.0649 (with lanthanum) and 0.0618 au dL mg–1 (without lanthanum). In addition, these materials presented a better percentage of immobilization (>90%), recovered activity, resistance to storage, and detection range than materials containing 4,7-[1-carboxymethyl-(imidazol-3-ium)]-2,1,3-benzothiadiazole chloride. Therefore, the combination of GO-BTD (Im/Ac)/ChOx (with or without lanthanum) affords efficient biosensors for the colorimetric detection of cholesterol.