The effect of support hydrophobicity on lipase activity and substrate selectivity was investigated with and without Triton X-100 (TX-100). Lipases from Thermomyces lanuginosa (TL) and Alcaligenes sp. (QLM) were immobilized on graphene oxide (GO) and a range of chemically reduced graphene oxides (CRGOs) with different levels of surface hydrophobicity. Activity assays using 4-hydroxy-N-propyl-1,8-naphthalimide (NAP) esters of varying chain lengths (NAP-butyrate (NAP-B), NAP-octanoate (NAP-O), and NAP-palmitate (NAP-P)) showed that the activity of immobilized QLM and TL decreased by more than 60% on GO and 80% on CRGO (2 h), with activity decreasing further as surface hydrophobicity of the CRGOs increased. Across the hydrophobicity range of GO/CRGOs, the substrate selectivity of QLM shifted from more readily hydrolyzing NAP-P to NAP-B, while TL retained its substrate selectivity for NAP-O. Lipase TL was also shown to desorb from GO and 2 h CRGO when mixed with NAP-O and NAP-P, whereas QLM did not. Circular dichroism analyses of the lipase α-helix content correlate to the observed activity data, with decreases in the α-helical content (40% in TL and 20% in QLM relative to free lipase) consistent with decreases in activity after immobilization on GO. α-Helical content decreased even further as the surface hydrophobicity of CRGOs increased. Attenuated total reflectance-Fourier transform infrared spectroscopy also showed significant changes to the lipase secondary structure upon immobilization. The addition of TX-100 into the activity assay modified the substrate selectivity of immobilized QLM, improving the activity against NAP-O (90%) and NAP-P (67%) compared to the activity measured without TX-100. It was shown that TX-100 primarily affected the activity of QLM by interacting with the ester substrate and the lipase itself. This study provides an improved understanding of how support hydrophobicity and the presence of TX-100 can affect activity/selectivity of lipases immobilized on hydrophobic supports.