In situ and laboratory studies of sea—surface microlayer sampling methods using glass plate, rotating drum, screen and funnel samplers were conducted. For glass plate and rotating drum samplers, surface microlayer samples of different thickness were collected by controlling their withdrawal rate and rotating rate. The relationships between pH, surface tension, the concentration of dissolved trace metals Cu and Pb, phosphate, particulate matters and sampling thickness were carefully investigated. It was shown that physicochemical and biological properties change obviously at the sampling thickness of about 50 μm, which is consistent with the mean thickness of the boundary film in the models of gas exchange across the sea surface. It is proposed that the apparent sampling thickness of the surface microlayer should be less than 40 μm. The factors affecting the sampling thickness are discussed, and the feasibility and applicable conditions for different sampling methods are evaluated.