Optical methods for life sciences is a very comprehensive subject. Especially in this era, scientific discoveries depend on more and more interdisciplinary cooperation. For example, the discovery of fluorescent proteins and probes helps optical engineers achieve higher contrast and localization images for biological samples. Optical diffraction limitation was eliminated by combining reversible fluorescent probes and super-resolution microscopy. In addition, by means of a novel light-sensitive channel-rhodopsin or voltage sensor, we can generate noninvasive and accurate light to activate or inhibit target neurons for brain function study. In pathology diagnosis, endoscopes serving as very important tools for the surgeon were developed to be much more miniature and higher resolution for the patients. Of course, innovative imaging modalities such as photoacoustic imaging, spectroscopy, and optical coherence tomography (OCT) were also developed for deeper and noninvasive tissue diagnosis. This special issue introduces some new progress in optical methods for life sciences.