In the past two decades, two-photon microscopy (TPM) transforms biomedical research, allowing nondestructive high-resolution fluorescent molecular imaging and label-free imaging in vivo and in real time. Here we review the recent advances of TPM technology including novel laser sources, new image acquisition paradigms, and microendoscopic imaging systems. Then, we survey the capabilities of TPM imaging of biological relevant molecules such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and reactive oxygen species (ROS). Biomedical applications of TPM in neuroscience and cancer detection are demonstrated.