The construction of synthetic enzymatic reaction networks can provide new insights into the design principles of living systems. However, the programmable connection of enzymes into a wide range of network topologies has been challenging due to the lack of a general strategy enabling a reversible activity regulation of individual network enzymes. Here, we exploit a general and modular strategy based on the external regulation of enzymes using light and photoswitchable inhibitors (PIs) that enables the bottom-up construction and control of enzymatic systems studied under out-of-equilibrium conditions. Upon synthesis and incorporation of potent photoswitchable trypsin inhibitors (Tr-PIs), the output of several functional enzymatic systems could be photoregulated using 390/460 nm light as a trigger signal. In addition, the wavelength-selective control over the activity of two enzymes within a functional bi-enzymatic system was achieved using a suitable combination of two PIs.