Electron-beam lithography employing poly(tert-Buthyl- Methacrylate)-co-(Methyl-Methacrylate) as radiation sensitive system was used to pattern bioactive molecules at super-high resolution. Positive and negative tone lithography succeed in printing fluorescent avidin into the range of 100 nm resolution. Two mechanisms were used for protein attachment, namely: (1) the linkage of the amino-end of the protein to the radiation-induced carboxylic acid sites, via NH2-to-COOH crosslinking mediated by carbodiimide; and (2) hydrophobic interaction between the patterned proteins and unexposed surfaces, in contrast to hydrophilic-repulsive interaction with exposed one. The first mechanism produces positive tone, half-tone images, while the second produces negative tone, sharp contrast images. On this basis, we assume that the first mechanism is concentration-controlled, while the second is an on-off one. This study proves that e-beam lithography materials and techniques can be easily transferred in bio- microlithography, with impact on biodevices fabrication and combinatorial chemistry.