Anaerobic biosynthetic crystals (ANBC) were prepared based on microbially induced calcium precipitation (MICP) and their potential explored for groundwater defluoridation and decalcification. The preparation conditions of ANBC were optimized and the influence of key factors (initial fluoride ions (F-) concentration, pH, and initial calcium ions (Ca2+) concentration) on the crystals was investigated. During the operation of the reactor, at pH of 7.0, the hydraulic retention time (HRT) of 6 h, and Ca2+ concentration of 180mg L-1, a maximum removal efficiency reached 93.31%, while 66.20% of Ca2+ could be removed. The adsorption dynamics study showed that the adsorption of ANBC was most in line with the pseudo-second-order model. The stability of ANBC operation was studied and failure reaction showed that the crystals maintained a stable removal ability after 35 times of repeated use. Further studies found that this was attributed to the continuous growth and synthesis of the crystals. The defluoridation and decalcification mechanism was further explored by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and X-ray diffraction spectra (XRD). This study innovatively proposes a method for biosynthesis of crystals under anaerobic conditions based on MICP, which can efficiently and stably remove F- and Ca2+ in groundwater, providing a valuable strategy for groundwater contaminant remediation and energy saving.