This invention provides a novel monomeric insulin (B27K-DTrI, B27K-destripeptide-Insulin), its composition and the method of preparation therefore. Highly pure B27K-DTrI-Insulin is monomeric (non-associative at high concentration and physiological pH) with in-vivo bioactivity being 80% of that of native insulin. B27K-DTrI-Insulin can be produced by enzyme cleavage of a monomeric insulin precursor secreted by yeast instead of by the less efficient enzyme transpeptidation as known in the prior art. The new method increases the total yield and is favorable for industrial production.