Hybrid imaging technology has the potential to provide reliable imaging and accurate detection of cancer cells by combining the advantages and overcoming the shortages of various clinical imaging tools. Nanomaterials with unique targeting properties and their small size have improved biomedical imaging. Indeed, their small size determines local contrast agent concentrations in tumors by enhanced permeability and retention (EPR) effect. In this work, amino-modified silica-coated Gadolinium-Copper Nanoclusters were fabricated and conjugated to AS1411 aptamer (Apt-ASGCuNCs) and radiolabeled with technetium-99 m (^99mTc) for in vivo fluorescence imaging, magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT). The synthesized nanoconjugate was fully characterized by transmission electron microscopy (TEM), element mapping, fluorescence spectroscopy, and Fourier-transform infrared spectroscopy. Moreover, XTT assay, and apoptosis and necrosis methods were applied to study toxicity. Radiochemical yield was calculated 93% that revealed a great potential for complex formation between Apt-ASGCuNCs and ^99mTcO₄⁻. Also, good stability of ^99mTc-Apt-ASGCuNCs was found in the human serum up to 4 h. Both Apt-ASGCuNCs and ^99mTc-Apt-ASGCuNCs indicated a considerable tumor-targeting in in vivo fluorescence imaging, MRI and SPECT with 4T1 tumor-bearing BALB/c mice. The biodistribution results showed no undesirable accumulation of ^99mTc-Apt-ASGCuNCs in the liver, and spleen as it circulated freely in the blood pool. Meanwhile, ^99mTc-Apt-ASGCuNCs were removed from the body through the renal clearance system, making it more convenient for future multimodality imaging applications.