Molecularly imprinted micro-solid phase extraction fiber was developed by modifying molecularly imprinted polymer film on the surface of silica fiber exploring “grafting via surface attached monomer” (method I) and “grafting via sol–gel” (method II) approaches. The latter approach was found to be inferior to the former one in terms of the sensitivity of insulin detection [method I, LOD=0.009ngmL−1; method II, LOD=0.064ngmL−1, RSD=1.21%]. Notably, either of the techniques, molecularly imprinted micro-solid phase extraction or complementary sensor, was found to be incompetent to monitor the stringent level of insulin in the real samples. However, the combination of these techniques has been found quite suitable for achieving the high detection sensitivity of ultra-trace insulin in human blood serum and Huminsulin injection, without any non-specific (false-positives) contributions. The proposed hyphenated device could serve as a possible marker for risk of developing type 2 diabetes mellitus and diabetic coma due to insulin resistance in human beings.