The stable operation of partial nitrification and anammox (PNA) process is a challenge in nitrogen removal from extremely low-strength ammonia wastewater like sewage mainstream. A moving bed reactor with functional carriers (30% filling rate) was developed to treat a synthetic influent with 50 mg/L ammonia. The long-term operation results showed nitrogen removal efficiencies of 71.7 ± 9.1% have been stably obtained under a relatively short hydraulic retention time of 2 h. Microbial analysis revealed anammox bacteria and ammonium oxidizing bacteria (AOB) with 29.7% and 6.32% abundance were the two most dominant bacteria in the reactor. Carriers largely retained slow-growing anammox bacteria in their hollow space and established a sandwich-like biofilm structure of co-immobilization of anammox bacteria and AOB. The anammox activity was much higher in carrier biofilms than in suspended flocs, while, for the AOB activity, the situation was reversed. Correspondingly, a fluorescent in situ hybridization analysis illustrated the active cell fractions of anammox bacteria and AOB in carrier biofilms were 63.7% and 4.8%, and 2.7% and 61.4% in suspended flocs. Biofilm formation and dissolved oxygen control were deemed to be the two key factors affecting the optimal co-immobilization of anammox bacteria and AOB, which guaranteed the efficient PNA.