A remote sensing technique was developed to detect citrus canker in laboratory conditions and was verified in the grove by utilizing an unmanned aerial vehicle (UAV). In the laboratory, a hyperspectral (400–1000 nm) imaging system was utilized for the detection of citrus canker in several disease development stages (i.e., asymptomatic, early, and late symptoms) on Sugar Belle leaves and immature (green) fruit by using two classification methods: (i) radial basis function (RBF) and (ii) K nearest neighbor (KNN). The same imaging system mounted on an UAV was used to detect citrus canker on tree canopies in the orchard. The overall classification accuracy of the RBF was higher (94%, 96%, and 100%) than the KNN method (94%, 95%, and 96%) for detecting canker in leaves. Among the 31 studied vegetation indices, the water index (WI) and the Modified Chlorophyll Absorption in Reflectance Index (ARI and TCARI 1) more accurately detected canker in laboratory and in orchard conditions, respectively. Immature fruit was not a reliable tissue for early detection of canker. However, the proposed technique successfully distinguished the late stage canker-infected fruit with 92% classification accuracy. The UAV-based technique achieved 100% classification accuracy for identifying healthy and canker-infected trees.