Plasma-based sterilization at atmospheric pressure in plasma needle configuration is a recently developing technique that has numerous applications in heat-sensitive materials and enhances biosensor properties. In this study, the capacitively coupled He–O2/Ar and He–N2/Ar mixtures [Formula: see text]-plasma at atmospheric pressure is characterized to find out the optimum condition of plasma-based sterilization for plasma needle. Trace rare gas actinometry based on optical emission spectroscopy has been used to estimate the [O] and [N] atomic densities as a function of discharge parameters i.e., applied pulsing voltage, gases concentration and different flow rates. Ar-1line Ar[Formula: see text] at 750 nm, N-I line [Formula: see text] at 746.8 nm and O-I line [Formula: see text] at 844.6 nm is used to determine the atomic densities of oxygen [O] and nitrogen [N]. Using the Boltzmann plot, the rotational temperature of He–N2/Ar and He–O2/Ar was calculated from the rotational levels of the “first negative system” (FNS) [Formula: see text]. The temperature of the gas increases as the pulsed DC power increases, but it drops as the gas flow rate in the mixture increases. The actinometric results show that both densities increase with applied pulsing voltage and molecular concentration of oxygen and nitrogen. Moreover, it is also observed that emission intensity ratios ([Formula: see text] and [Formula: see text]) also increase with an increase in applied pulsing voltage but they decrease with an increase in flow rate. Based on these results, it is suggested that the optimum conditions, in terms of [O] and [N] atomic densities, for plasma-based sterilization in needle configuration are 4% oxygen and nitrogen concentration at 4 kV applied pulsing voltage and 100[Formula: see text]SCCM flow rate.