Betanin is a natural pigment with significant antioxidant and biological activities currently used as food colorant. The isolation of betanin is problematic due to its instability. In this work, we developed a fast and economic procedure based on molecularly imprinted solid-phase extraction (MISPE) for the selective clean-up of betanin and its stereoisomer isobetanin from beetroot extracts. Dipicolinic acid was used as template for the MIP preparation because of its structural similarity with the chromophore group of betanin. The MISPE procedures were fully optimized allowing the almost complete removal of matrix components such as sugars and proteins, resulting in high extraction recovery of betanin/isobetanin in a single step. Moreover, the whole extraction procedure was performed in environmentally friendly solvents with either ethanol or water. Our MISPE method is very promising for the future development of well-formulated beetroot extract with specified betanin/isobetanin content, ready for food or medicinal use.