This paper presents the development of taper-in-taper fiber (TITF)–optical fiber-based sensors for chemical and biological detection. TITF structure is a fiber structure formed by fabricating a taper again on the taper region of the traditional taper fiber. The experimental results show that the TITF structure has a lower transmitted intensity than the common taper fiber structure. It is demonstrated that the structure of TITF is more conducive to evanescent waves, thereby making it more sensitive to changes in the refractive index (RI) of the external environment. To confirm this, measurements of ethanol solutions with different water contents are taken using the TITF structure to showcase the chemical sensing, thus verifying its sensitivity to RI changes in the external environment. The sensor can measure ethanol solutions from 0 to 100% with a sensitivity of 4.06 a.u./%. Thereafter, the TITF sensor is modified with ZnO-NPs to enhance its sensitivity for biological sensing (creatinine detection). The transmitted intensity of different concentrations of creatinine solution (0–2000 μM) is measured using the proposed sensor. The sensor showed good linearity and a high sensitivity of 0.11 a.u./μM for creatinine solution. Therefore, it is demonstrated that the TITF sensor with ZnO-NPs can be used for effective biological sensing.