This study systematically analyzed reactive species generated with self-built helium-based lowerature atmospheric-pressure plasma jet (He-APPJ); we measured the hydroxyl radical (•OH) and hydrogen peroxide (H2O2) above surfaces of plasma-treating solutions and in plasma-treated medium (PTM) of different solutions, including deionized water, phosphate buffered saline, and Dulbecco's modified Eagle medium. The effects of adding O2 into He working gas on •OH and H2O2 generation were discussed. The MCF7 cell responses to plasma treatment were also observed. The •OH emission (309 nm) in He-APPJ and above surfaces of plasma-treating solutions were analyzed through optical emission spectroscopy, and concentrations of •OH and H2O2 in different solutions were measured using terephthalic acid and Amplex Red, respectively. In our plasma system, it was observed that higher conductivity of solutions may cause stronger •OH emission above solution. The ingredients of the solution may affect the concentrations of •OH and H2O2. Addition of 0.1% O2 generates strongest •OH emission above surfaces of plasma-treating solution with our He-APPJ, and thus, the highest H2O2 concentration in PTM. The MCF7 cell viability is influenced by the H2O2 concentration in PTM. The results of this study can facilitate further study of plasma effects on PTM and cell culture.