We compared the fluorescence (λex = 355 nm, λem = 450 nm) of chromophore‐containing dissolved organic matter (CDOM) determined in the laboratory to that determined with a shipboard fluorometer and an airborne laser fluorosensor off the U.S. mid‐Atlantic coast. Fluorescence was highly correlated with CDOM absorption at the excitation wavelength despite the presence of multiple sources of CDOM and a changing contribution of CDOM to the total dissolved organic C (DOC) pool, both in surface waters and down the water column. The mean quantum yield (0.8±0.1%) fell within the narrow range of yields previously determined for U.S. east coast and south Florida waters. These results show that CDOM absorption can be reliably determined from in situ or airborne fluorescence over wide areas of the ocean and that it may be possible to estimate DOC concentration from fluorescence or absorption measurements.