The dotted matrix structures on Ti6Al4V specimens with different spacing were manufactured by laser technology. Four kinds of self-assembled monolayers (SAMs) were prepared on the above Ti6Al4V substrates by self-assembled technique. The surface characteristics of Ti6Al4V specimens were characterized with topography and contact angle measurement. The results show that the water contact angles of the Ti6Al4V specimens increase significantly by dotted matrix structure manufacturing and SAMs deposition. The contact angles of the Ti6Al4V specimens deposited with long chain molecules of FDTS, FOTS and OTS are larger than 150° and the superhydrophobic surfaces are formed. The maximum contact angle of 164.5° can be acquired when FDTS SAMs are deposited on the Ti6Al4V specimens. The superhydrophobic surface of the Ti6Al4V specimens can be formed for short chain MPS molecule deposition only when the spacing of laser manufacturing is set to 50 μm. The superhydrophobic surface will change to the hydrophobic surface with spacing increasing. The contact angles of the Ti6Al4V specimens for all four SAMs deposition reduce with increasing of the spacing of dotted matrix