Highly sensitive and cost effective measurement tools are required in biotechnology research and applications. Fluorescence provides very simple, cost effective, and sensitive methods in most of the biosensor techniques. Spectrometer is an essential tool for any kind of spectroscopic measurements. A charged coupled device (CCD)-based fiber optic spectrometer is highly compact, light weight, and an extremely easy to use tool. In this chapter, we have described the use of CCD-based fiber-optic spectrometers in detection of fluorescence signal from a fiber-optic-based sensor. The method can easily be extended to fluorescence detection in any other application.