Continuous recording of the dynamic changes of multiple physiologically neurochemicals in vivo is vital to understand the molecular basis of brain functions. For instance, disentangling the complicated interrelationship between ascorbic acid (AA) and copper ions (Cu2+) in signal transduction and homeostasis remains essential in understanding the pathology of hypoxia damage and neurodegenerative diseases. Unfortunately, to the best of our knowledge, there is still no report regarding the development of online and in vivo electrochemical or optical methods that can simultaneously measure AA and Cu2+ due to the limitation of different detection mechanisms and environmental conditions. In this work, we report a parallel dual-channel online optical detection platform (OODP) for the continuous and simultaneous monitoring of AA and Cu2+ in a living rat brain by integrating a capillary-based microfluidic system as well as an optical detector built under a bright-field microscope. Two kinds of colorimetric sensors, oxidized tetramethylbenzidine (oxTMB) and dopamine functionalized silver nanoparticles (DA-AgNPs), were used to recognize AA and Cu2+ in physically isolated detection channels. Combined with in vivo microdialysis sampling, the dual-channel OODP exhibited a wide linearity for AA and Cu2+ detection in the ranges of 0.5 μM to 100 μM and 0.1 μM to 10 μM, respectively. Compared to online electrochemical systems, this dual-channel OODP shows the following advantages: (1) isolated detection positions for two species, which can easily avoid crosstalk, and (2) a small dead-volume of 0.0113 μL in the detector, which obviously improves the time resolution. Along with its high stability, selectivity, and a near real-time response, the constructed dual-channel OODP can be successfully used to monitor cerebral AA and Cu2+ alterations simultaneously in a living rat brain.