A new spectrophotometric reagent 2-Hydroxy-1-napthaldehyde-orthoaminophenol has been synthesized and characterized. A very simple, ultra-sensitive, highly selective and non-extractive new spectrophotometric method for the simultaneous determination of arsenic (III) and arsenic (V) at nano-trace levels using 2-Hydroxy-1-napthaldehyde-orthoaminophenol (HNA-OAP)has been developed. HNA-OAP has been proposed as a new analytical reagent for the direct non-extractive spectrophotometric determination of arsenic (III&V). This novel spectrophotometric reagent reacts in a slightly acidic (0.00016-0.0004 M H2SO4) aqueous solution with arsenic(III) in 20% N,N-dimethylformamide (DMF) to produce highly absorbent red chelate with has an absorption maximum at 505 nm. The absorbance intensity of the metal-chelate reaches a constant value (after heating for 5 min at (45±5) 0C within 15 min and remains stable over 24 h. The average molar absorption coefficient and Sandell’s sensitivity were found to be 2.46×104 L mol-1cm-1 and 5-ng cm-2. Linear calibration graphs were obtained for 0.01 – 60-mgL-1 of As, having a detection limit of 1-ngmL-1; the quantification limit of the reaction system was found to be 10-ngmL-1 and the RSD was 0-3%. The stoichiometric composition of the chelate is 3:2 (As: HNA-OAP). A large excess of over 60 cations, anions and complexion agents (like, chloride, phosphate, azide, tartrate, oxalate, SCN- etc.) do not interfere in the determination. The developed method was successfully used in the determination of total arsenic in several certified reference materials (alloys, steels, ores, human urine, hair, nails, bovine liver and sediments) as well as in some biological fluids (human blood, urine, hair, nail and milk), soil samples, food samples (vegetables, fruits, rice, corn and wheat), solutions containing both arsenic(III) and arsenic(V) speciation and complex synthetic mixtures. The results of the proposed method for assessing groundwater, biological, food and soil samples were comparable with both ICP-OES & AHG-AAS and were found to be in excellent agreement. The method has high precision and accuracy (s = ± 0.01 for 0.5 mg L-1).