Fluorescence cross-correlation spectroscopy (FCCS) is a method that measures the temporal fluorescence fluctuations coming from two differently labeled molecules diffusing through a small sample volume. Cross-correlation analysis of the fluorescence signals from separate detection channels extracts information of the dynamics of the dual-labeled molecules. FCCS has become an essential tool for the characterization of diffusion coefficients, binding constants, kinetic rates of binding, and determining molecular interactions in solutions and cells. By cross-correlating between two focal spots, flow properties could also be measured. Recent developments in FCCS have been targeted at using different experimental schemes to improve on the sensitivity and address their limitations such as cross-talk and alignment issues. This review presents an overview of the different excitation and detection methodologies used in FCCS and their biological applications. This is followed by a description of the fluorescent probes currently available for the different methods. This will introduce biological readers to FCCS and its related techniques and provide a starting point to selecting which experimental scheme is suitable for their type of biological study.