We analyze the impact of the refractive index of optical fibers on the focusing properties of a miniaturized graded‐index (GRIN) fiber probe. The ABCD ray transfer matrix and characteristic parameters are employed to characterize the working distance, focusing spot size, and the depth of field effectively. The three‐dimensional (3‐D) function diagrams and two‐dimensional (2‐D) graphs are used to illustrate the impact of the no‐core fiber (NCF) refractive index and the center refractive index of GRIN fiber on the focusing properties. Numerical analysis results show that when the length of the NCF is 0.36 mm and the GRIN fiber is 0.1 mm, the variation of the refractive index of the NCF between 1.44 and 1.52 leads to the variation of the working distance in the range of 0.02 mm, while the focusing spot size varies in the range of 5 μm. A comparison of 12 probe samples reveals that a 0.02 difference in the center refractive index of GRIN fiber could result in a 0.16 mm variation in working distance and a variation in focusing spot size of over 3 μm. The experimental results indicate that alterations in the length of the fibers have a considerable effect on the focusing properties of the probe. In contrast, the range of variation in focusing properties with fiber refractive index is relatively limited.