The three-dimensional real-space observation of photonic nanojet-induced modes in a chain of microspheres with different diameters is reported. The optical transmission properties of a chain of microspheres are studied by using high resolution finite-difference time-domain calculation. The photonic nanojet-induced modes in different chains of microspheres are measured by using a scanning optical microscope system with an optical-fiber probe. We observe the photonic nanojet-induced modes from optical microscope images for chains of 3 μm, 5 μm, and 8 μm microspheres deposited on a patterned silicon substrate. The incident beam can be periodically reproduced in chains of dielectric microspheres giving rise to lossless periodically optical focusing with period of two diameters. Detailed theoretical and experimental data on the transmission, scattering loss, and field-of-view are presented. This waveguide technique can be used in biomedical microscopy, ultra-precise laser process, microfluidics, and nanophotonic circuits.