We report an annealing method which significantly enhances the piezoelectric performance by simultaneously tuning morphology and crystalline phase of electrospun P(VDF-TrFE) nanofibers. Annealing process-induced transformation, from α to β crystalline phase at lattice scale, also changes fiber morphology at micrometer scale. The underlying differences between as-spun and annealed P(VDF-TrFE) nanofibers for their stretch-hold deformation were examined via in-situ synchrotron for both small and wide angle X-ray scattering (SAXS and WAXS). Transmission X-ray microscopy (TXM) characterization reveals the underneath fiber orientation subjected to different levels of deformation. Multi-length scale microstructural evolution demonstrates distinct deformation behaviors of the as-spun and annealed nanofibers during uniaxial tensile loading. The findings can be applied for multi-scale structure modulation for the design of future piezoelectric devices.