Abstract: The present invention is a novel, ultra-small tip, internal referenced, amperometric microbiosensor that uses an immobilized biological interface to measure the concentration of an analyte in a specimen. It consists of a casing that narrows to an aperture having a diameter at the tip no greater than 4 .mu.m; enclosed within the casing a reference electrode and a working electrode both immersed in electrolyte; within the aperture, an inner polymer film, an immobilized biological interface layer, and an outer specimen-compatible, non-virulent polymer film. Another important feature of the present invention is that the microbiosensor can readily be encased in a durable protective sheath. The microbiosensor is especially useful in situ for specimens that cannot be mixed, such as in situ compounds in unmixed fluid, or semi-solid specimens.