This study aimed to enhance the adhesion strength between electroplated Cu and a low-profile Ajinomoto build-up film (ABF) substrate through isothermal annealing treatment. We examined the electroplated Cu microstructure evolution (including grain size, orientation, and texture) and elemental distribution at the Cu/ABF interface after annealing at 180 °C and 210 °C by means of electron backscatter diffraction (EBSD), X-ray diffraction (XRD), and transmission electron microscopy (TEM) in conjunction with energy dispersive X-ray spectroscopy (EDS). Furthermore, the adhesion strength of the Cu/ABF structure after isothermal annealing for different times was investigated through a peeling test based on the IPC-TM-650 test methods. The chemical bond and atomic concentration depth profiles of the fracture surface (ABF side) after the peeling test were characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). These investigations demonstrated that the adhesion strength of the Cu/ABF structure can be greatly enhanced by the electroplated Cu microstructure modification and Cu/ABF interdiffusion through appropriate isothermal annealing treatment.