Lung cancer is the most common causes of cancer-related deaths worldwide, and a lack of effective methodsfor early diagnosis has greatly impacted the prognosis and survival rates of the affected patients. Tumor-initiatingcells (TICs) are considered to be largely responsible for tumor genesis, resistance to tumor therapy, metastasis,and recurrence. In addition to representing a good potential treatment target, TICs can provide clues for the earlydiagnosis of cancer. MicroRNA (miRNA) alterations are known to be involved in the initiation and progressionof human cancer, and the detection of related miRNAs in TICs is an important strategy for lung cancer earlydiagnosis. As Hsa-miR-155 (miR-155) can be used as a diagnostic marker for non-small cell lung cancer (NSCLC),a smart molecular beacon of miR-155 was designed to image the expression of miR-155 in NSCLC cases. TICsexpressing CD133 and CD338 were obtained from A549 cells by applying an immune magnetic bead isolationsystem, and miR-155 was detected using laser-scanning confocal microscopy. We found that intracellular miR-155 could be successfully detected using smart miR-155 molecular beacons. Expression was higher in TICsthan in A549 cells, indicating that miR-155 may play an important role in regulating bio-behavior of TICs. Asa non-invasive approach, molecular beacons could be implemented with molecular imaging to diagnose lungcancer at early stages.