The bio-optical properties of coral reef waters were examined across coral reef ecosystems not influenced by land-derived run-off, in the Great Barrier Reef lagoon (Heron Island) and the Coral Sea (the Coringa-Herald and Lihou Reefs). The aim was to determine whether the absorption properties, the concentration-specific absorption properties, and the phytoplankton and non-algal pigmented particle (NAP) absorption concentrations varied from the ocean waters flushing onto the reef at high tide to those waters on the reef or flushing off the reef at low tide. The optical and biogeochemical properties of on-reef waters systematically differed from the surrounding ocean waters. The chl a concentration values varied up to 7-fold and the NAP concentrations up to 29-fold; for the reef samples, the chl a values were on average 2 to 3 times lower than for the oceans whilst the NAP values were slightly higher on the reefs. The spectral absorption values of the chl a, NAP, and colored dissolved organic matter (CDOM) varied up to 6-fold for reef waters and up to 15-fold for ocean waters. The spectral absorption for chl a was up to 3-fold lower on the reef waters, the absorption by the CDOM was up to 2-fold higher and the NAP absorption was 1.6-fold higher on the reef waters. The concentration-specific absorption coefficients for chl a and NAP varied up to 9-fold in reef waters and up to 30-fold in ocean waters. In the case of Heron Island and Coringa-Herald cays, this concentration-specific absorption was on average 1.3 to 1.7-fold higher for chl a and up to 2-fold lower for NAP on the reefs. The Lihou Reef measurements were more ambiguous between the reef waters and ocean waters due to the complex nature and size of this reef. Based on our results, the assumption that the optical properties of on-reef waters and the adjacent ocean waters are the same was shown to be invalid. Ocean waters flowing on to the reef are higher in phytoplankton, whilst waters on the reef or flowing off the reefs are higher in CDOM and NAP. We found differences in the pico,- nano-, and microplankton distributions as well as in the ratios of photosynthetic to photoprotective pigments. The variability in the bio-optical properties between the reef waters and adjacent ocean waters has implications for the estimations of sunlight absorption along the water column, the UV penetration depth, the temperature distributions, and the nutrient and carbon fluxes in coral reef ecosystems. As Earth observation algorithms require proper parameterization for the water column effects when estimating benthic cover, the actual optical properties need to be used. These results will improve the use of Earth observation to systematically map the differences in the water quality between reefs and the adjacent ocean.