Poor sensitivity and low specificity of current molecular imaging probes limit their application in clinical settings. To address these challenges, we used a process known as cell-SELEX to develop unique molecular probes termed aptamers with the high binding affinity, sensitivity, and specificity needed for in vivo molecular imaging inside living animals. Importantly, aptamers can be selected by cell-SELEX to recognize target cells, or even surface membrane proteins, without requiring prior molecular signature information. As a result, we are able to present the first report of aptamers molecularly engineered with signaling molecules and optimized for the fluorescence imaging of specific tumor cells inside a mouse. Using a Cy5-labeled aptamer TD05 (Cy5-TD05) as the probe, the in vivo efficacy of aptamer-based molecular imaging in Ramos (B-cell lymphoma) xenograft nude mice was tested. After intravenous injection of Cy5-TD05 into mice bearing grafted tumors, noninvasive, whole-body fluorescence imaging then allowed the spatial and temporal distribution to be directly monitored. Our results demonstrate that the aptamers could effectively recognize tumors with high sensitivity and specificity, thus establishing the efficacy of these fluorescent aptamers for diagnostic applications and in vivo studies requiring real-time molecular imaging.