Alkanethiol anchored self-assembled monolayers (SAMs) on gold are widely used to immobilize and detect molecules including DNA and proteins. Most of these molecules are covalently bonded with the SAM on the Au surface and cannot be released easily. By using different functional groups, the interfacial charge of SAMs can be selected, and thus, they can be considered as adaptors for immobilizing and releasing materials selectively through electrostatic interaction under given conditions. In this work, as an additional factor to control the surface charge, SAMs with mixed functional groups are presented, and it is demonstrated that the isoelectric point (IEP) can be tailored by the ratio of functional groups. Using carboxylic acid- and amine-SAM on gold substrates as an example, isoelectric points (IEPs) from 3.5 to 6.5 can be obtained arbitrarily. The ratio between the functional groups on the surface was quantified by X-ray photoelectron spectrometry (XPS) and was found to be slightly different from the deposition solution. The homogeneous spatial distribution of the functional groups was determined with scanning electrical potential microscopy (SEPM). The interfacial charge of SAMs with mixed functional groups on gold was investigated by electrokinetic analysis in aqueous electrolyte solutions.