This paper describes laser-based methods for preparing micropatterns of bioactive molecular species in self-assembled monolayers (SAMs) and micropatterns of proteins and other biological molecules immobilized on solid substrates. Applications of these micropatterned surfaces in multianalyte biosensing and tissue engineering are emphasized. The focus of the paper is on the use of a computer-controlled laser ablation system comprising a research-grade inverted optical microscope, a pulsed nitrogen-pumped dye laser emitting at 390 nm, a programmable sample stage, and the computerized control system. The laser system can be implemented in a typical biosensor or tissue culture laboratory to enable the facile and reproducible fabrication of micropatterned surfaces by several methods. Various methods for patterning are discussed with examples given and emphasis placed on (1) laser ablation in the fabrication of photolithography masks, (2) electrochemical patterning of SAMs, and (3) laser desorption of SAMs. The relative merits of each technique are discussed with respect to application in fabrication of active surfaces for biosensing and tissue culture applications.