In this work, a self-assembled surface-enhanced Raman scattering (SERS) sensor based on an optofluidic microstructured hollow fiber (MHF) integrated composites of graphene oxide (GO), silver nanoparticles (Ag NPs), and 4-mercaptophenylboronic acid (4-MPBA) as a glucose detection device was proposed. This SERS substrate is specific to glucose and grows on the outside of the core of MHF. Here, the MHF has a microchannel, where a suspended core was attached. In the microchannel, the trace sample solution was interacted with the SERS substrate, realizing the Raman online sensing detection of trace cerebrospinal fluid glucose. In the range of 0.5-10 mmol/L, the results present that detection of the glucose concentration shows good linearity without external interference. Meanwhile, this type of rapid, online, and label-free SERS sensor of glucose shows the ability to detect excessive glucose content in the actual cerebrospinal fluid environment of the Sprague-Dawley rats, providing a simple detection method for meningitis, meningeal multiple malignant tumor metastasis, and related diseases caused by abnormal glucose content in cerebrospinal fluid.