Layered multi-materials of dissimilar polymers and their nanocomposites offer new opportunities as smart materials and structures. A critical aspect of such structures is the quality of interlayer adhesion between dissimilar polymer matrices. This work reports the development of asymmetric double cantilever beam (ADCB) specimens of dissimilar polymers and its use in the analysis and understanding of their interlayer adhesion in 3D-printed rigid/soft interfaces. Acrylonitrile butadiene styrene (ABS), polycarbonate (PC), polylactic acid (PLA) were chosen as the rigid polymers and combined with thermoplastic polyurethane (TPU) as the soft component. 3D-printed ADCB specimens were loaded under opening mode, until fracture, to obtain the load-displacement data and the fracture surfaces were analyzed using optical microscopy. ABS/TPU/ABS and PC/TPU/PC material combinations resulted in a more stable crack growth with a high peak load indicating a relatively good interfacial adhesion. The high nozzle temperatures of ABS and PC and their amorphous nature contributed to a good layer-to-layer fusion during 3D printing. However, PLA/TPU/PLA specimens exhibited an unstable crack growth behavior with a pure adhesive failure mode and a significantly lower peak load. This poor interfacial bond strength was correlated to the relatively low nozzle temperature of PLA and its semi-crystalline structure. The maximum loads in ABS/TPU/ABS and PC/TPU/PC specimens were found to be ∼2.5 times greater than that of PLA/TPU/PLA ones. The method provides a valuable tool in quantifying interlayer adhesion quality in printed dissimilar polymers and their functional nanocomposites.