The capacity of biochar to take up heavy metals from contaminated soil and water is influenced by the pyrolysis temperature. We have prepared three biochar samples from Jerusalem artichoke stalks (JAS) by pyrolysis at 300, 500 and 700 °C, denoted as JAS300, JAS500, and JAS700, respectively. A variety of synchrotron-based techniques were used to assess the effect of pyrolysis temperature on the molecular properties and copper (Cu) sorption capacity of the samples. The content of oxygen-containing functional groups in the biochar samples decreased, while that of aromatic structures and alkaline mineral components increased, with a rise in pyrolysis temperature. Scanning transmission X-ray microscopy indicated that sorbed Cu(II) was partially reduced to Cu(I), but this process was more evident with JAS300 and JAS700 than with JAS500. Carbon K-edge X-ray absorption near edge structure spectroscopy indicated that Cu(II) cations were sorbed to biochar via complexation and Cu-π bonding. With rising pyrolysis temperature, Cu(II)-complexation weakened while Cu-π bonding was enhanced. In addition, the relatively high ash content and pH of JAS500 and JAS700 facilitated Cu precipitation and the formation of langite on the surface of biochar. The results of this investigation will aid the conversion of halophyte waste to useable biochar for the effective remediation of Cu-contaminated soil and water.