Laser focusing is an important parameter that affects the characteristics of laser-induced plasma. Focusing lenses with different F-numbers form different energy density distributions near the surface of a sample, thus affecting the characteristics of plasma. In this study, the plasma generated by a nanosecond laser ablation of a micro-alloy steel certified sample at 1 atm of air was investigated. We compare the spectrally integrated plasma images obtained at different defocusing distances for short- and long-focus lenses and investigate the optical emission spectra of laser-induced plasma on steel alloy by using focusing lenses with different F-numbers. With an increase in the defocusing distance, the plasma plume changes from flat to hemispherical and then splitting occurs. The spectral line intensity increases first and then decreases, then increases slightly, and finally decreases gradually. For the long-focus lens, when the focal point is above the sample surface, the laser beam strongly interacts with air over a longer distance, leading to longer air plasma and weaker sample plasma compared with the short-focus lens. Thus, the relative intensity of the second peak in the spectral line intensity, according to the defocusing distance, gradually decreases with increasing F-number. We also obtain two-dimensional spatial distributions of the spectral line intensity according to the F-number and defocusing distance. The optimal defocusing distances for all focusing lenses occur when the focal point is below the sample surface. The relation between the optimal defocusing distance and F-number follows a single- exponential decay function.