Biomarkers are indicator systems that help to understand the state of health of the patients with high accuracy and help to prevent the disease spread by control measures. Incorporating a biorecognition unit into the molecular ensemble provides quick and powerful ways to detect and distinguish target analytes from diverse samples. It is crucial to identify suitable biomarkers for better clinical intervention and treatment at the early stages of a disease. Optical sensors with image-based technologies have extra advantages such as high sensitivity, reliability, robustness, and potential to be combined on optoelectronic devices. Optical fluorescence imaging provides cellular and subcellular resolutions and image enhancement in 3D and real-time. Fluorescence-based imaging is used in a broad variety of preclinical investigations, and there is great interest in finding further ways to translate the principle into clinical applications. This chapter focuses on the current progress in the use of different fluorescent indicators for the different types of cancer biomarker detection and provides a comprehensive overview of their principles, methodologies, and applications. In addition, we discuss ongoing issues and limitations of the existing systems and methods. Further, we present the key requirements of portable biosensors and the perspective of optical biosensors for cancer biomarker detection.