Three-dimensional (3D) control over the placement of bioactive cues is fundamental to understand cell guidance and develop engineered tissues. Two-photon patterning (2PP) provides such placement at micro- to millimeter scale, but nonspecific interactions between proteins and functionalized extracellular matrices (ECMs) restrict its use. Here, a 2PP system based on nonfouling hydrophilic photocages and Sortase A (SA)-based enzymatic coupling is presented, which offers unprecedented orthogonality and signal-to-noise ratio in both inert hydrogels and complex mammalian matrices. Improved photocaged peptide synthesis and protein functionalization protocols with broad applicability are introduced. Importantly, the method enables 2PP in a single step in the presence of fragile biomolecules and cells, and is compatible with time-controlled growth factor presentation. As a corollary, the guidance of axons through 3D-patterned nerve growth factor (NGF) within brain-mimetic ECMs is demonstrated. The approach allows for the interrogation of the role of complex signaling molecules in 3D matrices, thus helping to better understand biological guidance in tissue development and regeneration.