Background/Objectives: Antimicrobial resistance (AMR) surveillance is a cornerstone of national AMR strategies but requires sustained, cross-sectoral financing. While the need for such financing is well recognized, its quantification remains scarce in low- and middle-income countries. This study aimed to estimate the full costs of AMR surveillance across the human health, animal health, and food sectors (2021–2030) in selected facilities in Nepal and generate evidence to inform sustainable financing. Methods: A bottom-up micro-costing approach was used to analyze data from five sites. Costs were adjusted for inflation using projected gross domestic product deflators, and probabilistic sensitivity analyses were conducted to assess uncertainty in laboratory sample volumes under four scenarios. Results: The total cost of AMR surveillance in Nepal was $6.7 million: $3.4 million for human health (50.3% out of the aggregated costs), $2.7 million for animal health (39.8%), and $0.7 million for the food sector (9.9%). Laboratories accounted for >90% of total costs, with consumables and personnel as the main cost drivers. Average cost per sample was $150 (animal), $64 (food), and $6 (human). Conclusions: This study offers the first robust, multi-sectoral 10-year cost estimates of AMR surveillance in Nepal. The findings highlight that sustaining AMR surveillance requires predictable domestic financing, particularly to cover recurrent laboratory operations as donor support declines. These results provide cost evidence to support future budgeting and policy planning toward sustainable, nationally financed AMR surveillance in Nepal.