By means of a slow drying process and the control of surface charge characteristics, protein stripe patterns were readily prepared on the luminal surface of a capillary. We systematically studied the effects of surface properties, pH, and protein concentration on pattern formation using optical microscopy, atomic force microscopy, and quartz crystal microbalance measurement. By balancing these parameters, a broad selection of proteins could be assembled within a capillary with well-defined stripe patterns. Neutravidin, one of the model proteins, was specifically chosen to demonstrate the bioactivity retained through the assembly process by interaction with fluorescently labeled biotin motifs. This technique therefore offers a facile approach for patterning proteins and other biomacromolecules in capillary tubes.