In wastewater treatment, an alternative to the widely used aerobic nitrification with subsequent anoxic denitrification method is the combination of nitration and anammox (AMX) in one system. This study focuses on the co-immobilization of AMX and ammonia-oxidizing bacteria into a polyvinyl alcohol (PVA) hydrogel, and its effective use in nitrogen removal (NR). The NR process was performed in nine consecutive, repeated batches. By optimizing the conditions of the biotransformations, there was equal utilization of nitrogen in both sources, N–NH4+ and N–NO2−, at 100% NR during the sixth repetition. A significant increase in the immobilized co-culture activity was also detected per cycle. The maximum value of the NR rate was 3.46 mg N (L h)−1, and 100% NR efficiency was achieved with an initial concentration of 100.3 mg N L−1 for N–NH4+ and 60.1 mg N L−1 for N–NO2−, during the eighth batch biotransformation.