Theranostic nanomedicine was successfully prepared using layered double hydroxide (LDH). By means of step-by-step incorporation of an anticancer drug, methotrexate (MTX), and a radioisotope, Co-57, into the interlayer space and lattice of LDH, respectively, theranostic hybrid Nanomedicine could be prepared. X-ray diffractometry, scanning electron microscopy, X-ray adsorption spectroscopy, and Fourier-transform infrared spectroscopy systematically showed that incorporating the Co2+ into the MTX-LDH did not alter the crystalline phase, size, morphology, or intact structure of the hybrid. The labeled Co-57 in MTX-LDH was highly stable in human serum, showing almost 90% retention after 48 h. In vitro cellular uptake of Co-57-labeled MTX-LDH was very high in mouse colon carcinoma CT-26 cells, with ~60 ID% at 4 h. The cytotoxicity assay of MTX-LDH showed high cancer-cell suppression on CT-26 cells. To evaluate the diagnostic ability of Co-57-labeled MTX-LDH, in vivo single-photon emission computed tomography (SPECT) images were investigated on CT-26 xenografted mouse model. The SPECT signal in tumor tissue began to appear within 1 h, and it increased for 3 h.