Fluorescence lifetime measurements are a powerful tool in biomedical research and advances in detection technology make them ideally suited for the study of biomolecular interactions. Time-resolved techniques, compared to more conventional methods, provide improved precision and contrast in the monitoring of complex biological processes. Fluorescence lifetimes are extracted by using time-correlated single-photon counting, which offers single photon sensitivity, high temporal resolution and excellent signal to noise ratio. Furthermore, combining this technique with microfluidics offers unprecedented advantages. For example, in analytical applications, apart from the high sensitivity required, the study of analytes often demands low sample consumption and short mixing times to allow for the monitoring of quick reactions. These parameters can nicely be achieved with the use of microfluidics. Hydrodynamic focusing within 3-inlet 1-outlet continuous flow microfluidic devices can be used as a molecular confinement mechanism to improve the detection efficiency as well as a means to enhance mixing within microchannels for the study of fast reaction kinetics. In this work, a powerful combination of confocal microscopy and microfluidics was used to perform fluorescence lifetime measurements on freely diffusing and freely flowing molecules. For this purpose, a home-built scanning confocal system was developed to ensure sufficient reduction in background levels, enabling the detection of fluorescence signal that arises from single molecules. Fluorescence lifetime imaging along with a maximum likelihood estimator adapted from single molecule studies was performed to visualise hydrodynamic focusing and characterise mixing within microfluidic devices. Time-resolved methods were also employed to detect single molecules freely flowing within microchannels. A novel fluorescence lifetime approach was developed to perform Förster resonance energy transfer measurements on freely diffusing molecules and subsequently applied for the study of streptavidin-biotin binding and protein conformational changes upon unfolding.