Monolithic immobilized pH gradient (M-IPG) materials were prepared in microchannles by photoinitiated polymerization of acrylamide, glycidylmethacrylate and Bis, followed by the attachment of focused Ampholine onto the surface of porous monoliths via epoxide groups. With M-IPG materials as matrix, FITC-labeled ribonuclease B, myoglobin and alpha-casein were well separated by microchip isoelectric focusing (mu CIEF) without carrier amphocytes (CAs) added in the buffer. Both chemical and pressure mobilization were applied to drive focused zones for LIF detection. Our experimental results showed that pressure mobilization was preferable with neglectable band broadening, and good peak shape and high detection sensitivity were obtained. All these results demonstrate that mu CIEF with M-IPG materials is not only an efficient mode for protein enrichment and separation but also attractive to couple with other CE modes to achieve multi-dimensional separation or MS for further identification, without the interference of mobile CAs.