<p>Biological silk is a natural optical waveguide with humidity-sensitive properties. The refractive index (RI) is a critical parameter that determines the optical properties of waveguides and is greatly affected by humidity. In this article, we propose an optical method for real-time measuring the RI of micron-scale biological silks at different humidity. We employ a section of single-mode fiber (SMF) and a section of biological silk to configure an F-P cavity structure. By recording the spectrum and diameter of the silk under different humidity, we can obtain the relationship among free spectral range (FSR)、diameter, and relative humidity (RH). Based on this relationship, we calculate the variation of RI with humidity. We measure three materials in the 30–80% RH range. The measurement results indicate that during this process, the RI of spider egg sac silk (SESS) ranges from 1.491 ± 0.003 to 1.412 ± 0.009, mulberry silk is 1.551 ± 0.009 to 1.473 ± 0.005, and radial silk is 1.549 ± 0.005 to 1.479 ± 0.003. The relative uncertainty in the range ± 5 × 10^− 3 to ± 7 × 10^− 3 is achieved for these challenging samples. This rapid and convenient measurement method provides a new perspective for applying biological silks.</p>