Near infrared fluorescence (NIRF) spectroscopy is capable of sensitive and selective detection of semiconductive, single-walled carbon nanotubes (SWNT) using the unique electronic bandgap properties of these carbon allotropes. We reported here the first detection and quantitation of SWNT in sediment and biota at environmentally relevant concentrations using NIRF spectroscopy. In addition, we utilized this technique to qualitatively characterize SWNT samples before and after ecotoxicity, bioavailability and fate studies in the aquatic environment. Sample preparation prior to NIRF analysis consisted of surfactant-assisted high power ultrasonication. The bile salt sodium deoxycholate (SDC) enabled efficient extraction and disaggregation of SWNT prior to NIRF analysis. The method was validated using standard-addition experiments in two types of estuarine sediments, yielding recoveries between 66 ± 7% and 103 ± 10% depending on SWNT type and coating used, demonstrating the ability to isolate SWNT from complex sediment matrices. Instrument detection limits were determined to be 15 ng mL–1 SWNT in 2% SDC solution and method detection limits (including a concentration step) were 62 ng g–1 for estuarine sediment, and 1.0 μg L–1 for water. Our work has shown that NIRF spectroscopy is highly sensitive and selective for SWNT and that this technique can be applied to track the environmental and biological fate of this important class of carbon nanomaterial in the aquatic environment.