The properties and stability of the reported surface nanobubbles are related to the substrate used and the generation method. Here, we design a series of experiments to study the influence of the hydrophobicity of the substrate and the production method on the formation and properties of nanobubbles. We choose three different substrates, dodecyltrichlorosilane (DTS) modified silicon, octadecyltrichlorosilane (OTS) modified silicon, and highly oriented pyrolytic graphite (HOPG) as nanobubble substrates, and two methods of ethanol–water exchange and 4-C cold water to produce nanobubbles. It is found that using ethanol-water exchange method could produce more and larger nanobubbles than the 4-C cold water method. The contact angle of nanobubbles produced by ethanol–water exchange depends on the hydrophobicity of substrates, and decreases with the increase of the hydrophobicity of substrates. More interestingly, nanoscopic contact angle approaches the macroscopic contact angle as the hydrophobicity of substrates increases. It is believed that these results would be very useful to understand the stability of surface nanobubbles.